Human G-protein coupled receptors

ABSTRACT

Human G-protein coupled receptor polypeptides and DNA (RNA) encoding such polypeptides and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptides for identifying antagonists and agonists to such polypeptides and methods of using the agonists and antagonists therapeutically to treat conditions related to the underexpression and overexpression of the G-protein coupled receptor polypeptides, respectively. Also disclosed are diagnostic methods for detecting a mutation in the G-protein coupled receptor nucleic acid sequences and an altered level of the soluble form of the receptors.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation of U.S. patentapplication Ser. No. 08/465,973, filed Jun. 6, 1995, which disclosure isherein incorporated by reference; said 08/465,083is acontinuation-in-part of PCT/US95/04079, filed Mar. 30, 1995, whichdisclosure is herein incorporated by reference.

[0002] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as well as the production of suchpolynucleotides and polypeptides. More particularly, the polypeptides ofthe present invention are human 7-transmembrane receptors. Thetransmembrane receptors are G-protein coupled receptors sometimeshereinafter referred to individually as GPR1, GPR2, GPR3 and GPR4. Theinvention also relates to inhibiting the action of such polypeptides.

[0003] It is well established that many medically significant biologicalprocesses are mediated by proteins participating in signal transductionpathways that involve G-proteins and/or second messengers, e.g., cAMP(Lefkowitz, Nature, 351: 353-354 (1991)). Herein these proteins arereferred to as proteins participating in pathways with G-proteins or PPGproteins. Some examples of these proteins include the GPC receptors,such as those for adrenergic agents and dopamine (Kobilka, B. K., etal., PNAS, 84: 46-50 (1987); Kobilka, B. K., et al., Science, 238:650-656 (1987); Bunzow, J. R., et al., Nature, 336: 783-787 (1988)),G-proteins themselves, effector proteins, e.g., phospholipase C, adenylcyclase, and phosphodiesterase, and actuator proteins, e.g., proteinkinase A and protein kinase C (Simon, M. I., et al., Science, 252: 802-8(1991)).

[0004] For example, in one form of signal transduction, the effect ofhormone binding is activation of an enzyme, adenylate cyclase, insidethe cell. Enzyme activation by hormones is dependent on the presence ofthe nucleotide GTP, and GTP also influences hormone binding. A G-proteinconnects the hormone receptors to adenylate cyclase. G-protein was shownto exchange GTP for bound GDP when activated by hormone receptors. TheGTP-carrying form then binds to an activated adenylate cyclase.Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns theG-protein to its basal, inactive form. Thus, the G-protein serves a dualrole, as an intermediate that relays the signal from receptor toeffector, and as a clock that controls the duration of the signal.

[0005] The membrane protein gene superfamily of G-protein coupledreceptors has been characterized as having seven putative transmembranedomains. The domains are believed to represent transmembrane α-helicesconnected by extracellular or cytoplasmic loops. G-protein coupledreceptors include a wide range of biologically active receptors, such ashormone, viral, growth factor and neuroreceptors.

[0006] G-protein coupled receptors have been characterized as includingthese seven conserved hydrophobic stretches of about 20 to 30 aminoacids, connecting at least eight divergent hydrophilic loops. TheG-protein family of coupled receptors includes dopamine receptors whichbind to neuroleptic drugs used for treating psychotic and neurologicaldisorders. Other examples of members of this family include calcitonin,adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine,serotonin, histamine, thrombin, kinin, follicle stimulating hormone,opsins and rhodopsins, odorant, cytomegalovirus receptors, etc.

[0007] Most G-protein coupled receptors have single conserved cysteineresidues in each of the first two extracellular loops which formdisulfide bonds that are believed to stabilize functional proteinstructure. The 7 transmembrane regions are designated as TM1, TM2, TM3,TM4, TM5, TM6, and TM7. TM3 is also implicated in signal transduction.

[0008] Phosphorylation and lipidation (palmitylation or farnesylation)of cysteine residues can influence signal transduction of some G-proteincoupled receptors. Most G-protein coupled receptors contain potentialphosphorylation sites within the third cytoplasmic loop and/or thecarboxy terminus. For several G-protein coupled receptors, such as theβ-adrenoreceptor, phosphorylation by protein kinase A and/or specificreceptor kinases mediates receptor desensitization.

[0009] The ligand binding sites of G-protein coupled receptors arebelieved to comprise a hydrophilic socket formed by several G-proteincoupled receptors transmembrane domains, which socket is surrounded byhydrophobic residues of the G-protein coupled receptors. The hydrophilicside of each G-protein coupled receptor transmembrane helix ispostulated to face inward and form the polar ligand binding site. TM3has been implicated in several G-protein coupled receptors as having aligand binding site, such as including the TM3 aspartate residue.Additionally, TM5 serines, a TM6 asparagine and TM6 or TM7phenylalanines or tyrosines are also implicated in ligand binding.

[0010] G-protein coupled receptors can be intracellularly coupled byheterotrimeric G-proteins to various intracellular enzymes, ion channelsand transporters (see, Johnson et al., Endoc., Rev., 10: 317-331(1989)). Different G-protein α-subunits preferentially stimulateparticular effectors to modulate various biological functions in a cell.Phosphorylation of cytoplasmic residues of G-protein coupled receptorshave been identified as an important mechanism for the regulation ofG-protein coupling of some G-protein coupled receptors.

[0011] G-protein coupled receptors are found in numerous sites within amammalian host, for example, dopamine is a critical neurotransmitter inthe central nervous system and is a G-protein coupled receptor ligand.

[0012] In accordance with one aspect of the present invention, there areprovided novel polypeptides which have been putatively identified asG-protein coupled receptors and biologically active and diagnosticallyor therapeutically useful fragments and derivatives thereof. Thepolypeptides of the present invention are of human origin.

[0013] In accordance with another aspect of the present invention, thereare provided isolated nucleic acid molecules encoding human G-proteincoupled receptors, including mRNAs, DNAs, cDNAs, genomic DNA as well asantisense analogs thereof and biologically active and diagnostically ortherapeutically useful fragments thereof.

[0014] In accordance with a further aspect of the present invention,there is provided a process for producing such polypeptides byrecombinant techniques which comprises culturing recombinant prokaryoticand/or eukaryotic host cells, containing a human G-protein coupledreceptor nucleic acid sequence, under conditions promoting expression ofsaid protein and subsequent recovery of said protein.

[0015] In accordance with yet a further aspect of the present invention,there are provided antibodies against such polypeptides.

[0016] In accordance with another embodiment, there is provided aprocess for using the receptors to screen for receptor antagonistsand/or agonists and/or receptor ligands.

[0017] In accordance with still another embodiment of the presentinvention there is provided a process of using such agonists tostimulate the G-protein coupled receptors for the treatment ofconditions related to the under-expression of the G-protein coupledreceptors.

[0018] In accordance with another aspect of the present invention thereis provided a process of using such antagonists for inhibiting theaction of the G-protein coupled receptors for treating conditionsassociated with over-expression of the G-protein coupled receptors.

[0019] In accordance with yet another aspect of the present inventionthere is provided non-naturally occurring synthetic, isolated and/orrecombinant G-protein coupled receptor polypeptides which are fragments,consensus fragments and/or sequences having conservative amino acidsubstitutions, of at least one transmembrane domain of the G-proteincoupled receptor, such that G-protein coupled receptor polypeptides ofthe present invention may bind G-protein coupled receptor ligands, orwhich may also modulate, quantitatively or qualitatively, G-proteincoupled receptor ligand binding.

[0020] In accordance with still another aspect of the present inventionthere are provided synthetic or recombinant G-protein coupled receptorpolypeptides, conservative substitution and derivatives thereof,antibodies, anti-idiotype antibodies, compositions and methods that canbe useful as potential modulators of G-protein coupled receptorfunction, by binding to ligands or modulating ligand binding, due totheir expected biological properties, which may be used in diagnostic,therapeutic and/or research applications.

[0021] It is still another object of the present invention to providesynthetic, isolated or recombinant polypeptides which are designed toinhibit or mimic various G-protein coupled receptors or fragmentsthereof, as receptor types and subtypes.

[0022] In accordance with yet a further aspect of the present invention,there is also provided diagnostic probes comprising nucleic acidmolecules of sufficient length to specifically hybridize to theG-protein coupled receptor nucleic acid sequences.

[0023] In accordance with yet another object of the present invention,there is provided a diagnostic assay for detecting a disease orsusceptibility to a disease related to a mutation in a G-protein coupledreceptor nucleic acid sequence.

[0024] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

[0025] The following drawings are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

[0026] FIGS. 1-4 show the cDNA sequences and the corresponding deducedamino acid sequences of the four G-protein coupled receptors of thepresent invention, respectively. The standard one-letter abbreviationfor amino acids are used. Sequencing was performed using a 373 AutomatedDNA sequencer (Applied Biosystems, Inc.). Sequencing accuracy ispredicted to be greater than 97% accurate.

[0027]FIG. 5 is an illustration of the amino acid homology between GPR1(top line) and odorant receptor-like protein (bottom line).

[0028]FIG. 6 illustrates the amino acid homology between GPR2 (top line)and the human Endothelial Differentiation Gene-1 (EDG-1) (bottom line).

[0029]FIG. 7 illustrates the amino acid homology between GPR3 (top line)and a human G-protein coupled receptor open reading frame (ORF) (bottomline).

[0030]FIG. 8 illustrates the amino acid homology between GPR4 and thechick orphan G-protein coupled receptor (bottom line).

[0031] In accordance with an aspect of the present invention, there areprovided isolated nucleic acids (polynucleotides) which encode for themature polypeptides having the deduced amino acid sequences of FIGS. 1-4(SEQ ID No. 2, 4, 6 and 8) or for the mature polypeptides encoded by thecDNAs of the clones deposited as ATCC Deposit No. 75981 (GPR1), 75983(GPR2), 75976 (GPR3), 75979 (GPR4) on Dec. 16, 1994.

[0032] A polynucleotide encoding the GPR1 polypeptide of the presentinvention may be isolated from the human breast. The polynucleotideencoding GPR1 was discovered in a cDNA library derived from humaneight-week-old embryo. It is structurally related to the Gprotein-coupled receptor family. It contains an open reading frameencoding a protein of 296 amino acid residues. The protein exhibits thehighest degree of homology to an odorant receptor-like protein with 66%identity and 83% similarity over a 216 amino acid stretch.

[0033] A polynucleotide encoding the GPR2 polypeptide of the presentinvention may be isolated from human liver, heart and leukocytes. Thepolynucleotide encoding GPR2 was discovered in a cDNA library derivedfrom human adrenal gland tumor. It is structurally related to the Gprotein-coupled receptor family. It contains an open reading frameencoding a protein of 393 amino acid residues. The protein exhibits thehighest degree of homology to human EDG-1 with 30% identity and 52%similarity over a 383 amino acid stretch. Potential ligands to GPR2include but are not limited to anandamide, serotonin, adrenalin andnoradrenalin.

[0034] A polynucleotide encoding the GPR3 polypeptide of the presentinvention may be isolated from human liver, kidney and pancreas. Thepolynucleotide encoding GPR3 was discovered in a cDNA library derivedfrom human neutrophil. It is structurally related to the Gprotein-coupled receptor family. It contains an open reading frameencoding a protein of 293 amino acid residues. The protein exhibits thehighest degree of homology to a human G-Protein Coupled Receptor openreading frame with 39% identity and 61% similarity over the entire aminoacid sequence. Potential ligands to GPR3 include but are not limited toplatelet activating factor, thrombin, C5a and bradykinin.

[0035] A polynucleotide encoding the GPR4 polypeptide of the presentinvention may be found in human heart, spleen and leukocytes. Thepolynucleotide encoding GPR4 was discovered in a cDNA library derivedfrom human twelve-week-old embryo. It is structurally related to theG-protein coupled receptor family. It contains an open reading frameencoding a protein of 344 amino acid residues. The protein exhibits thehighest degree of homology to a chick orphan G-protein coupled receptorwith 82% identity and 91% similarity over a 291 amino acid stretch.Potential ligands to GPR4 include but are not limited to thrombin,chemokine, and platelet activating factor.

[0036] The polynucleotides of the present invention may be in the formof RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptides may beidentical to the coding sequence shown in FIGS. 1-4 (SEQ ID No. 1, 3, 5and 7) or that of the deposited clones or may be a different codingsequence which coding sequence, as a result of the redundancy ordegeneracy of the genetic code, encodes the same mature polypeptides asthe DNA of FIGS. 1-4 (SEQ ID No. 1, 3, 5 and 7) or the deposited cDNAs.

[0037] The polynucleotides which encode for the mature polypeptides ofFIGS. 1-4 (SEQ ID No. 2, 4, 6 and 8) or for the mature polypeptidesencoded by the deposited cDNAs may include: only the coding sequence forthe mature polypeptide; the coding sequence for the mature polypeptide(and optionally additional coding sequence) and non-coding sequence,such as introns or non-coding sequence 5′ and/or 3′ of the codingsequence for the mature polypeptide.

[0038] Thus, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes only coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

[0039] The present invention further relates to variants of thehereinabove described polynucleotides which encode for fragments,analogs and derivatives of the polypeptides having the deduced aminoacid sequence of FIGS. 1-4 (SEQ ID No. 2, 4, 6 and 8) or thepolypeptides encoded by the cDNAs of the deposited clones. The variantsof the polynucleotides may be a naturally occurring allelic variant ofthe polynucleotides or a non-naturally occurring variant of thepolynucleotides.

[0040] Thus, the present invention includes polynucleotides encoding thesame mature polypeptides as shown in FIGS. 1-4 (SEQ ID No. 2, 4, 6 and8) or the same mature polypeptides encoded by the cDNAs of the depositedclones as well as variants of such polynucleotides which variants encodefor a fragment, derivative or analog of the polypeptides of FIGS. 1-4(SEQ ID No. 2, 4, 6 and 8) or the polypeptides encoded by the cDNAs ofthe deposited clones. Such nucleotide variants include deletionvariants, substitution variants and addition or insertion variants.

[0041] As hereinabove indicated, the polynucleotides may have a codingsequence which is a naturally occurring allelic variant of the codingsequences shown in FIGS. 1-4 (SEQ ID No. 1, 3, 5 and 7) or of the codingsequences of the deposited clones. As known in the art, an allelicvariant is an alternate form of a polynucleotide sequence which may havea substitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptides.

[0042] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the polypeptides of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptides fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37: 767 (1984)).

[0043] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

[0044] Fragments of the full length gene of the present invention may beused as a hybridization probe for a cDNA library to isolate the fulllength cDNA and to isolate other cDNAs which have a high sequencesimilarity to the gene or similar biological activity. Probes of thistype preferably have at least 30 bases and may contain, for example, 50or more bases. The probe may also be used to identify a cDNA clonecorresponding to a full length transcript and a genomic clone or clonesthat contain the complete gene including regulatory and promotorregions, exons, and introns. An example of a screen comprises isolatingthe coding region of the gene by using the known DNA sequence tosynthesize an oligonucleotide probe. Labeled oligonucleotides having asequence complementary to that of the gene of the present invention areused to screen a library of human cDNA, genomic DNA or mRNA to determinewhich members of the library the probe hybridizes to.

[0045] The present invention further relates to polynucleotides whichhybridize to the hereinabove-described sequences if there is at least70%, preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIGS. 1, 2, 3 and 4 (SEQID NO: 1, 3, 5 and 7) or the deposited cDNA(s).

[0046] Alternatively, the polynucleotide may have at least 20 bases,preferably 30 bases, and more preferably at least 50 bases whichhybridize to a polynucleotide of the present invention and which has anidentity thereto, as hereinabove described, and which may or may notretain activity. For example, such polynucleotides may be employed asprobes for the polynucleotide of SEQ ID NO: 1, 3, 5 and 7 for example,for recovery of the polynucleotide or as a diagnostic probe or as a PCRprimer.

[0047] Thus, the present invention is directed to polynucleotides havingat least a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO: 2 as well as fragments thereof, which fragments have atleast 30 bases and preferably at least 50 bases and to polypeptidesencoded by such polynucleotides.

[0048] The deposit(s) referred to herein will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for purposes of Patent Procedure. Thesedeposits are provided merely as convenience to those of skill in the artand are not an admission that a deposit is required under 35 U.S.C.§112. The sequence of the polynucleotides contained in the depositedmaterials, as well as the amino acid sequence of the polypeptidesencoded thereby, are incorporated herein by reference and arecontrolling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

[0049] The present invention further relates to G-protein coupledreceptor polypeptides which have the deduced amino acid sequences ofFIGS. 1-4 (SEQ ID No. 2, 4, 6 and 8) or which have the amino acidsequences encoded by the deposited cDNAs, as well as fragments, analogsand derivatives of such polypeptides.

[0050] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptides of FIGS. 1-4 (SEQ ID No. 2, 4, 6 and 8) or that encodedby the deposited cDNAs, means a polypeptide which either retainssubstantially the same biological function or activity as suchpolypeptide, i.e. functions as a G-protein coupled receptor, or retainsthe ability to bind the ligand or the receptor even though thepolypeptide does not function as a G-protein coupled receptor, forexample, a soluble form of the receptor. An analog includes a proproteinwhich can be activated by cleavage of the proprotein portion to producean active mature polypeptide.

[0051] The polypeptides of the present invention may be recombinantpolypeptides, a natural polypeptides or synthetic polypeptides,preferably recombinant polypeptides.

[0052] The fragment, derivative or analog of the polypeptides of FIGS.1-4 (SEQ ID No. 2, 4, 6 and 8) or that encoded by the deposited cDNAsmay be (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide which isemployed for purification of the mature polypeptide. Such fragments,derivatives and analogs are deemed to be within the scope of thoseskilled in the art from the teachings herein.

[0053] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0054] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

[0055] The polypeptides of the present invention include the polypeptideof SEQ ID NO: 2, 4, 6 and 8 (in particular the mature polypeptide) aswell as polypeptides which have at least 70% similarity (preferably atleast 70% identity) to the polypeptide of SEQ ID NO: 2, 4, 6 and 8 andmore preferably at least 90% similarity (more preferably at least 90%identity) to the polypeptide of SEQ ID NO: 2, 4, 6 and 8 and still morepreferably at least 95% similarity (still more preferably at least 90%identity) to the polypeptide of SEQ ID NO: 2, 4, 6 and 8 and alsoinclude portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

[0056] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0057] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention. The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0058] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the G-protein coupled receptor genes. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

[0059] The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

[0060] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0061] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0062] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0063] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

[0064] As representative examples of appropriate hosts, there may bementioned: bacterial cells, such as E. coli, Streptomyces, Salmonellatyphimurium; fungal cells, such as yeast; insect cells such asDrosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowesmelanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

[0065] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: PWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, PSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

[0066] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0067] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

[0068] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0069] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), the disclosure of which is hereby incorporated byreference.

[0070] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0071] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences. Optionally, the heterologous sequence can encodea fusion protein including an N-terminal identification peptideimparting desired characteristics, e.g., stabilization or simplifiedpurification or expressed recombinant product.

[0072] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0073] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0074] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0075] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0076] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well know to those skilled in the art.

[0077] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23: 175 (1981), and other cell lines capable ofexpressing a compatible vector, for example, the C127, 3T3, CHO, HeLaand BHK cell lines. Mammalian expression vectors will comprise an originof replication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

[0078] The G-protein coupled receptor polypeptides can be recovered andpurified from recombinant cell cultures by methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Protein refolding steps can beused, as necessary, in completing configuration of the mature protein.Finally, high performance liquid chromatography (HPLC) can be employedfor final purification steps.

[0079] The polypeptides of the present invention may be a naturallypurified product, or a product of chemical synthetic procedures, orproduced by recombinant techniques from a prokaryotic or eukaryotic host(for example, by bacterial, yeast, higher plant, insect and mammaliancells in culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

[0080] Fragments of the full length G-protein coupled receptor genes maybe employed as a hybridization probe for a cDNA library to isolate thefull length genes and to isolate other genes which have a high sequencesimilarity to the gene or similar biological activity. Probes of thistype generally have at least 20 bases. Preferably, however, the probeshave at least 30 bases and may contain, for example, 50 bases or more.In many cases, the probe has from 20 to 50 bases. The probe may also beused to identify a cDNA clone corresponding to a full length transcriptand a genomic clone or clones that contain the complete G-proteincoupled receptor gene including regulatory and promotor regions, exons,and introns. As an example of a screen comprises isolating the codingregion of the G-protein coupled receptor gene by using the known DNAsequence to synthesize an oligonucleotide probe. Labeledoligonucleotides having a sequence complementary to that of the gene ofthe present invention are used to screen a library of human cDNA,genomic DNA or mRNA to determine which members of the library the probehybridizes to.

[0081] The G-protein coupled receptors of the present invention may beemployed in a process for screening for antagonists and/or agonists forthe receptor.

[0082] In general, such screening procedures involve providingappropriate cells which express the receptor on the surface thereof.Such cells include cells from mammals, yeast, drosophila or E. coli. Inparticular, a polynucleotide encoding the receptor of the presentinvention is employed to transfect cells to thereby express therespective G-protein coupled receptor. The expressed receptor is thencontacted with a test compound to observe binding, stimulation orinhibition of a functional response.

[0083] One such screening procedure involves the use of melanophoreswhich are transfected to express the respective G-protein coupledreceptor of the present invention. Such a screening technique isdescribed in PCT WO 92/01810 published Feb. 6, 1992.

[0084] Thus, for example, such assay may be employed for screening for areceptor antagonist by contacting the melanophore cells which encode theG-protein coupled receptor with both the receptor ligand and a compoundto be screened. Inhibition of the signal generated by the ligandindicates that a compound is a potential antagonist for the receptor,i.e., inhibits activation of the receptor.

[0085] The screen may be employed for determining an agonist bycontacting such cells with compounds to be screened and determiningwhether such compound generates a signal, i.e., activates the receptor.

[0086] Other screening techniques include the use of cells which expressthe G-protein coupled receptor (for example, transfected CHO cells) in asystem which measures extracellular pH changes caused by receptoractivation, for example, as described in Science, volume 246, pages181-296 (October 1989). For example, potential agonists or antagonistsmay be contacted with a cell which expresses the G-protein coupledreceptor and a second messenger response, e.g. signal transduction or pHchanges, may be measured to determine whether the potential agonist orantagonist is effective.

[0087] Another such screening technique involves introducing RNAencoding the G-protein coupled receptors into Xenopus oocytes totransiently express the receptor. The receptor oocytes may then becontacted in the case of antagonist screening with the receptor ligandand a compound to be screened, followed by detection of inhibition of acalcium signal.

[0088] Another screening technique involves expressing the G-proteincoupled receptors in which the receptor is linked to a phospholipase Cor D. As representative examples of such cells, there may be mentionedendothelial cells, smooth muscle cells, embryonic kidney cells, etc. Thescreening for an antagonist or agonist may be accomplished ashereinabove described by detecting activation of the receptor orinhibition of activation of the receptor from the phospholipase secondsignal.

[0089] Another method involves screening for antagonists by determininginhibition of binding of labeled ligand to cells which have the receptoron the surface thereof. Such a method involves transfecting a eukaryoticcell with DNA encoding the G-protein coupled receptor such that the cellexpresses the receptor on its surface and contacting the cell with apotential antagonist in the presence of a labeled form of a knownligand. The ligand can be labeled, e.g., by radioactivity. The amount oflabeled ligand bound to the receptors is measured, e.g., by measuringradioactivity of the receptors. If the potential antagonist binds to thereceptor as determined by a reduction of labeled ligand which binds tothe receptors, the binding of labeled ligand to the receptor isinhibited.

[0090] G-protein coupled receptors are ubiquitous in the mammalian hostand are responsible for many biological functions, including manypathologies. Accordingly, it is desirous to find compounds and drugswhich stimulate the G-protein coupled receptors on the one hand andwhich can antagonize a G-protein coupled receptor on the other hand,when it is desirable to inhibit the G-protein coupled receptor.

[0091] For example, agonists for G-protein coupled receptors may beemployed for therapeutic purposes, such as the treatment of asthma,Parkinson's disease, acute heart failure, hypotension, urinaryretention, and osteoporosis.

[0092] In general, antagonists to the G-protein coupled receptors may beemployed for a variety of therapeutic purposes, for example, for thetreatment of hypertension, angina pectoris, myocardial infarction,ulcers, asthma, allergies, benign prostatic hypertrophy and psychoticand neurological disorders, including schizophrenia, manic excitement,depression, delirium, dementia or severe mental retardation,dyskinesias, such as Huntington's disease or Gilles dila Tourett'ssyndrome, among others. G-protein coupled receptor antagonists have alsobeen useful in reversing endogenous anorexia and in the control ofbulimia.

[0093] Examples of G-protein coupled receptor antagonists include anantibody, or in some cases an oligopeptide, which binds to the G-proteincoupled receptors but does not elicit a second messenger response suchthat the activity of the G-protein coupled receptors is prevented.Antibodies include anti-idiotypic antibodies which recognize uniquedeterminants generally associated with the antigen-binding site of anantibody. Potential antagonists also include proteins which are closelyrelated to the ligand of the G-protein coupled receptors, i.e. afragment of the ligand, which have lost biological function and whenbinding to the G-protein coupled receptors, elicit no response.

[0094] A potential antagonist also includes an antisense constructprepared through the use of antisense technology. Antisense technologycan be used to control gene expression through triple-helix formation orantisense DNA or RNA, both of which methods are based on binding of apolynucleotide to DNA or RNA. For example, the 5′ coding portion of thepolynucleotide sequence, which encodes for the mature polypeptides ofthe present invention, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervanet al., Science, 251: 1360 (1991)), thereby preventing transcription andthe production of G-protein coupled receptors. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofmRNA molecules into G-protein coupled receptors (antisense—Okano, J.Neurochem., 56: 560 (1991); Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of G-protein coupled receptors.

[0095] Another potential antagonist is a small molecule which binds tothe G-protein coupled receptor, making it inaccessible to ligands suchthat normal biological activity is prevented. Examples of smallmolecules include but are not limited to small peptides or peptide-likemolecules.

[0096] Potential antagonists also include a soluble form of a G-proteincoupled receptor, e.g. a fragment of the receptors, which binds to theligand and prevents the ligand from interacting with membrane boundG-protein coupled receptors.

[0097] This invention additionally provides a method of treating anabnormal condition related to an excess of G-protein coupled receptoractivity which comprises administering to a subject the antagonist ashereinabove described along with a pharmaceutically acceptable carrierin an amount effective to block binding of ligands to the G-proteincoupled receptors and thereby alleviate the abnormal conditions.

[0098] The invention also provides a method of treating abnormalconditions related to an under-expression of G-protein coupled receptoractivity which comprises administering to a subject a therapeuticallyeffective amount of the agonist described above in combination with apharmaceutically acceptable carrier, in an amount effective to enhancebinding of ligands to the G-protein coupled receptor and therebyalleviate the abnormal conditions.

[0099] The soluble form of the G-protein coupled receptors, antagonistsand agonists may be employed in combination with a suitablepharmaceutical carrier. Such compositions comprise a therapeuticallyeffective amount of the antagonist or agonist, and a pharmaceuticallyacceptable carrier or excipient. Such a carrier includes but is notlimited to saline, buffered saline, dextrose, water, glycerol, ethanol,and combinations thereof. The formulation should suit the mode ofadministration.

[0100] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the pharmaceutical compositions may be employed in conjunctionwith other therapeutic compounds.

[0101] The pharmaceutical compositions may be administered in aconvenient manner such as by the topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes. Thepharmaceutical compositions are administered in an amount which iseffective for treating and/or prophylaxis of the specific indication. Ingeneral, the pharmaceutical compositions will be administered in anamount of at least about 10 μg/kg body weight and in most cases theywill be administered in an amount not in excess of about 8 mg/Kg bodyweight per day. In most cases, the dosage is from about 10 μg/kg toabout 1 mg/kg body weight daily, taking into account the routes ofadministration, symptoms, etc.

[0102] The G-protein coupled receptor polypeptides, and antagonists oragonists which are polypeptides, may be employed in accordance with thepresent invention by expression of such polypeptides in vivo, which isoften referred to as “gene therapy.”

[0103] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

[0104] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

[0105] Retroviruses from which the retroviral plasmid vectorshereinabove mentioned may be derived include, but are not limited to,Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0106] The vector includes one or more promoters. Suitable promoterswhich may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990(1989), or any other promoter (e.g., cellular promoters such aseukaryotic cellular promoters including, but not limited to, thehistone, pol III, and β-actin promoters). Other viral promoters whichmay be employed include, but are not limited to, adenovirus promoters,thymidine kinase (TK) promoters, and B19 parvovirus promoters. Theselection of a suitable promoter will be apparent to those skilled inthe art from the teachings contained herein.

[0107] The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

[0108] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein byreference in its entirety. The vector may transduce the packaging cellsthrough any means known in the art. Such means include, but are notlimited to, electroporation, the use of liposomes, and CaPO₄precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

[0109] The producer cell line generates infectious retroviral vectorparticles which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

[0110] The present invention also provides a method for determiningwhether a ligand not known to be capable of binding to a G-proteincoupled receptor can bind to such receptor which comprises contacting amammalian cell which expresses a G-protein coupled receptor with theligand under conditions permitting binding of ligands to the G-proteincoupled receptor, detecting the presence of a ligand which binds to thereceptor and thereby determining whether the ligand binds to theG-protein coupled receptor.

[0111] This invention further provides a method of screening drugs toidentify drugs which specifically interact with, and bind to, the humanG-protein coupled receptors on the surface of a cell which comprisescontacting a mammalian cell comprising an isolated DNA molecule encodingthe G-protein coupled receptor with a plurality of drugs, determiningthose drugs which bind to the mammalian cell, and thereby identifyingdrugs which specifically interact with and bind to a human G-proteincoupled receptor of the present invention.

[0112] This invention also provides a method of detecting expression ofthe G-protein coupled receptor on the surface of a cell by detecting thepresence of mRNA coding for a G-protein coupled receptor which comprisesobtaining total mRNA from the cell and contacting the mRNA so obtainedwith a nucleic acid probe comprising a nucleic acid molecule of at least15 nucleotides capable of specifically hybridizing with a sequenceincluded within the sequence of a nucleic acid molecule encoding a humanG-protein coupled receptor under hybridizing conditions, detecting thepresence of mRNA hybridized to the probe, and thereby detecting theexpression of the G-protein coupled receptor by the cell.

[0113] This invention is also related to the use of the G-proteincoupled receptor genes as part of a diagnostic assay for detectingdiseases or susceptibility to diseases related to the presence ofmutations in the G-protein coupled receptor genes. Such diseases, by wayof example, are related to cell transformation, such as tumors andcancers.

[0114] Individuals carrying mutations in the human G-protein coupledreceptor genes may be detected at the DNA level by a variety oftechniques. Nucleic acids for diagnosis may be obtained from a patient'scells, such as from blood, urine, saliva, tissue biopsy and autopsymaterial. The genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR (Saiki et al., Nature, 324: 163-166(1986)) prior to analysis. RNA or cDNA may also be used for the samepurpose. As an example, PCR primers complementary to the nucleic acidencoding the G-protein coupled receptor proteins can be used to identifyand analyze G-protein coupled receptor mutations. For example, deletionsand insertions can be detected by a change in size of the amplifiedproduct in comparison to the normal genotype. Point mutations can beidentified by hybridizing amplified DNA to radiolabeled G-proteincoupled receptor RNA or alternatively, radiolabeled G-protein coupledreceptor antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

[0115] Sequence differences between the reference gene and genes havingmutations may be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments may be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer isused with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

[0116] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230: 1242 (1985)).

[0117] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401(1985)).

[0118] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

[0119] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0120] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

[0121] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

[0122] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0123] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For a review of this technique, see Verma etal., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York (1988).

[0124] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0125] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0126] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0127] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0128] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0129] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,1975, Nature, 256: 495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies(Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96).

[0130] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice may be used to express humanized antibodies toimmunogenic polypeptide products of this invention.

[0131] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0132] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0133] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0134] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 50 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0135] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res., 8: 4057 (1980).

[0136] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0137] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., Id., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units to T4 DNAligase (“ligase”) per 0.5 μg of approximately equimolar amounts of theDNA fragments to be ligated.

[0138] Unless otherwise stated, transformation was performed asdescribed in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).

EXAMPLE 1 Bacterial Expression and Purification of GPR1

[0139] The DNA sequence encoding GPR1, ATCC # 75981, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ end sequences of the processed G-protein coupled receptor nucleotidesequence. Additional nucleotides corresponding to the GPR1 nucleotidesequence are added to the 5′ and 3′ sequences respectively. The 5′oligonucleotide primer has the sequence 5′GACTAAAGCTTAATGAGTAGTGAAATGGTG 3′ (SEQ ID No. 9) contains a HindIIIrestriction enzyme site followed by 19 nucleotides of G-protein coupledreceptor coding sequence starting from the presumed terminal amino acidof the processed protein. The 3′ sequence 5′GAACTTCTAGACCCTCAGGGTTGTAAATCAG 3′ (SEQ ID No. 10) containscomplementary sequences to an XbaI site and is followed by 20nucleotides of GPR1 coding sequence. The restriction enzyme sitescorrespond to the restriction enzyme sites on the bacterial expressionvector pQE-9 (Qiagen, Inc. Chatsworth, Calif.). pQE-9 encodes antibioticresistance (Amp^(r)), a bacterial origin of replication (ori), anIPTG-regulatable promoter operator (P/O), a ribosome binding site (RBS),a 6-His tag and restriction enzyme sites. pQE-9 is then digested withHindIII and XbaI. The amplified sequences are ligated into pQE-9 and areinserted in frame with the sequence encoding for the histidine tag andthe RBS. The ligation mixture is then used to transform E. coli strainM15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. etal., Molecular Cloning: A Laboratory Manual, Cold Spring LaboratoryPress, (1989). M15/rep4 contains multiple copies of the plasmid pREP4,which expresses the lacI repressor and also confers kanamycin resistance(Kan^(r)). Transformants are identified by their ability to grow on LBplates and ampicillin/kanamycin resistant colonies are selected. PlasmidDNA is isolated and confirmed by restriction analysis.

[0140] Clones containing the desired constructs are grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells are grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(“Isopropyl-B-D-thiogalacto pyranoside”) is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression. Cells are grownan extra 3 to 4 hours. Cells are then harvested by centrifugation. Thecell pellet is solubilized in the chaotropic agent 6 Molar GuanidineHCl. After clarification, solubilized G-protein coupled receptor ispurified from this solution by chromatography on a Nickel-Chelate columnunder conditions that allow for tight binding by proteins containing the6-His tag (Hochuli, E. et al., J. Chromatography 411: 177-184 (1984)).GPR1 is eluted from the column in 6 molar guanidine HCl pH 5.0 and forthe purpose of renaturation adjusted to 3 molar guanidine HCl, 100 mMsodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolarglutathione (oxidized). After incubation in this solution for 12 hoursthe protein is dialyzed to 10 mmolar sodium phosphate.

EXAMPLE 2

[0141] Bacterial Expression and Purification of GPR2

[0142] The DNA sequence encoding GPR2, ATCC # 75983, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ end sequences of the processed GPR2 coding sequence. Additionalnucleotides corresponding to GPR2 coding sequence are added to the 5′and 3′ sequences respectively. The 5′ oligonucleotide primer has thesequence 5′ GACTAAAGCTTAATGAGGCCCACATGGGCA 3′ (SEQ ID No. 11) contains aHindIII restriction enzyme site followed by 19 nucleotides of GPR2coding sequence starting from the presumed terminal amino acid of theprocessed protein. The 3′ sequence 5′ GAACTTCTAGACGAACTAGTGGATCCCCCCGG3′ (SEQ ID No. 12) contains complementary sequences to an XbaI site andis followed by 21 nucleotides of GPR2 coding sequence. The restrictionenzyme sites correspond to the restriction enzyme sites on the bacterialexpression vector pQE-9 (Qiagen, Inc. Chatsworth, Calif.). pQE-9 encodesantibiotic resistance (Amp^(r)), a bacterial origin of replication(ori), an IPTG-regulatable promoter operator (P/O), a ribosome bindingsite (RBS), a 6-His tag and restriction enzyme sites. pQE-9 is thendigested with HindIII and XbaI. The amplified sequences are ligated intopQE-9 and are inserted in frame with the sequence encoding for thehistidine tag and the RBS. The ligation mixture is then used totransform E. coli strain M15/rep 4 (Qiagen, Inc.) by the proceduredescribed in Sambrook, J. et al., Molecular Cloning: A LaboratoryManual, Cold Spring Laboratory Press, (1989). M15/rep4 contains multiplecopies of the plasmid pREP4, which expresses the laci repressor and alsoconfers kanamycin resistance (Kan^(r)). Transformants are identified bytheir ability to grow on LB plates and ampicillin/kanamycin resistantcolonies are selected. Plasmid DNA is isolated and confirmed byrestriction analysis.

[0143] Clones containing the desired constructs are grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells are grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(“Isopropyl-B-D-thiogalacto pyranoside”) is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression. Cells are grownan extra 3 to 4 hours. Cells are then harvested by centrifugation. Thecell pellet is solubilized in the chaotropic agent 6 Molar GuanidineHCl. After clarification, solubilized GPR2 is purified from thissolution by chromatography on a Nickel-Chelate column under conditionsthat allow for tight binding by proteins containing the 6-His tag(Hochuli, E. et al., J. Chromatography 411: 177-184 (1984)). GPR2 iseluted from the column in 6 molar guanidine HCl pH 5.0 and for thepurpose of renaturation adjusted to 3 molar guanidine HCl, 100 mM sodiumphosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione(oxidized). After incubation in this solution for 12 hours the proteinis dialyzed to 10 mmolar sodium phosphate.

EXAMPLE 3 Bacterial Expression and Purification of GPR3

[0144] The DNA sequence encoding GPR3, ATCC # 75976, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ end sequences of the processed G-protein coupled receptor nucleotidesequence. Additional nucleotides corresponding to the GPR3 codingsequence are added to the 5′ and 3′ sequences respectively. The 5′oligonucleotide primer has the sequence 5′GACTAAAGCTTAATGGCGTCTTTCTCTGCT 3′ (SEQ ID No. 13) contains a HindIIIrestriction enzyme site followed by 19 nucleotides of GPR3 codingsequence starting from the presumed terminal amino acid of the processedprotein. The 3′ sequence 5′ GAACTTCTAGACTTCACACAGTTGTACTAT 3′ (SEQ IDNo. 14) contains complementary sequences to XbaI site and is followed by19 nucleotides of GPR3 coding sequence. The restriction enzyme sitescorrespond to the restriction enzyme sites on the bacterial expressionvector pQE-9 (Qiagen, Inc. Chatsworth, Calif.). pQE-9 encodes antibioticresistance (Amp^(r)), a bacterial origin of replication (ori), anIPTG-regulatable promoter operator (P/O), a ribosome binding site (RBS),a 6-His tag and restriction enzyme sites. pQE-9 is then digested withXbaI and XbaI. The amplified sequences are ligated into pQE-9 and areinserted in frame with the sequence encoding for the histidine tag andthe RBS. The ligation mixture is then used to transform E. coli strainM15/rep 4 (Qiagen Inc.) by the procedure described in Sambrook, J. etal., Molecular Cloning: A Laboratory Manual, Cold Spring LaboratoryPress, (1989). M15/rep4 contains multiple copies of the plasmid pREP4,which expresses the lacI repressor and also confers kanamycin resistance(Kan^(r)). Transformants are identified by their ability to grow on LBplates and ampicillin/kanamycin resistant colonies are selected. PlasmidDNA is isolated and confirmed by restriction analysis.

[0145] Clones containing the desired constructs are grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells are grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(“Isopropyl-B-D-thiogalacto pyranoside”) is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression. Cells are grownan extra 3 to 4 hours. Cells are then harvested by centrifugation. Thecell pellet is solubilized in the chaotropic agent 6 Molar GuanidineHCl. After clarification, solubilized GPR3 is purified from thissolution by chromatography on a Nickel-Chelate column under conditionsthat allow for tight binding by proteins containing the 6-His tag(Hochuli, E. et al., J. Chromatography 411: 177-184 (1984)). GPR3 iseluted from the column in 6 molar guanidine HCl pH 5.0 and for thepurpose of renaturation adjusted to 3 molar guanidine HCl, 100 mM sodiumphosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione(oxidized). After incubation in this solution for 12 hours the proteinis dialyzed to 10 mmolar sodium phosphate.

EXAMPLE 4 Bacterial Expression and Purification of GPR4

[0146] The DNA sequence encoding GPR4, ATCC # 75979, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ end sequences of the processed GPR4 nucleotide sequence. Additionalnucleotides corresponding to the GPR4 coding sequence are added to the5′ and 3′ sequences respectively. The 5′ oligonucleotide primer has thesequence 5′ GACTAAAGCTTAATGGTAAGCGTTAACAGC 3′ (SEQ ID No. 15) contains aHindIII restriction enzyme site followed by 19 nucleotides of GPR4coding sequence starting from the presumed terminal amino acid of theprocessed protein. The 3′ sequence 5′ GAACTTCTAGACTTCAGGCAGCAGATTCATT 3′(SEQ ID No. 16) contains complementary sequences to XbaI site and isfollowed by 20 nucleotides of GPR4 coding sequence. The restrictionenzyme sites correspond to the restriction enzyme sites on the bacterialexpression vector pQE-9 (Qiagen, Inc. Chatsworth, Calif.). pQE-9 encodesantibiotic resistance (Amp^(r)), a bacterial origin of replication(ori), an IPTG-regulatable promoter operator (P/O), a ribosome bindingsite (RBS), a 6-His tag and restriction enzyme sites. pQE-9 is thendigested with HindIII and XbaI. The amplified sequences are ligated intopQE-9 and are inserted in frame with the sequence encoding for thehistidine tag and the RBS. The ligation mixture is then used totransform E. coli strain M15/rep 4 (Qiagen, Inc.) by the proceduredescribed in Sambrook, J. et al., Molecular Cloning: A LaboratoryManual, Cold Spring Laboratory Press, (1989). M15/rep4 contains multiplecopies of the plasmid pREP4, which expresses the lacI repressor and alsoconfers kanamycin resistance (Kan^(r)). Transformants are identified bytheir ability to grow on LB plates and ampicillin/kanamycin resistantcolonies are selected. Plasmid DNA is isolated and confirmed byrestriction analysis.

[0147] Clones containing the desired constructs are grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells are grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(“Isopropyl-B-D-thiogalacto pyranoside”) is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression. Cells are grownan extra 3 to 4 hours. Cells are then harvested by centrifugation. Thecell pellet is solubilized in the chaotropic agent 6 Molar GuanidineHCl. After clarification, solubilized GPR4 is purified from thissolution by chromatography on a Nickel-Chelate column under conditionsthat allow for tight binding by proteins containing the 6-His tag(Hochuli, E. et al., J. Chromatography 411: 177-184 (1984)). GPR4 iseluted from the column in 6 molar guanidine HCl pH 5.0 and for thepurpose of renaturation adjusted to 3 molar guanidine HCl, 100 mM sodiumphosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione(oxidized). After incubation in this solution for 12 hours the proteinis dialyzed to 10 mmolar sodium phosphate.

EXAMPLE 5 Expression of Recombinant GPR1 in COS Cells

[0148] The expression of plasmid, GPR1 HA is derived from a vectorpcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E. coli replication origin, 4) CMVpromoter followed by a polylinker region, a SV40 intron andpolyadenylation site. A DNA fragment encoding the entire GPR1 precursorand a HA tag fused in frame to its 3′ end is cloned into the polylinkerregion of the vector, therefore, the recombinant protein expression isdirected under the CMV promoter. The HA tag correspond to an epitopederived from the influenza hemagglutinin protein as previously described(I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R.Lerner, 1984, Cell 37, 767). The infusion of HA tag to the targetprotein allows easy detection of the recombinant protein with anantibody that recognizes the HA epitope.

[0149] The plasmid construction strategy is described as follows:

[0150] The DNA sequence encoding GPR1, ATCC # 75981, is constructed byPCR using two primers: the 5′ primer 5′GTCCAAGCTTGCCACCATGAGTAGTGAAATGGTG 3′ (SEQ ID No. 17) contains a HindIIIsite followed by 18 nucleotides of GPR1 coding sequence starting fromthe initiation codon; the 3′ sequence 5′CTAGCTCGAGTCAAGCGTAGTCTGGGACGTCGTATGGGTAGCAGGGTTGTAAATCAGG 3′ (SEQ IDNo. 18) contains complementary sequences to an XhoI site, translationstop codon, HA tag and the last 15 nucleotides of the GPR1 codingsequence (not including the stop codon). Therefore, the PCR productcontains a HindIII site, GPR1 coding sequence followed by HA tag fusedin frame, a translation termination stop codon next to the HA tag, andan XhoI site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp,are digested with HindIII and XhoI restriction enzymes and ligated. Theligation mixture is transformed into E. coli strain SURE (StratageneCloning Systems, La Jolla, Calif.) the transformed culture is plated onampicillin media plates and resistant colonies are selected. Plasmid DNAis isolated from transformants and examined by restriction analysis forthe presence of the correct fragment. For expression of the recombinantGPR1, COS cells are transfected with the expression vector byDEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). Theexpression of the GPR1 HA protein is detected by radiolabeling andimmunoprecipitation method (E. Harlow, D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelledfor 8 hours with ³⁵S-cysteine two days post transfection. Culture mediaare then collected and cells are lysed with detergent (RIPA buffer (150mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5).(Wilson, I. et al., Id. 37: 767 (1984)). Both cell lysate and culturemedia are precipitated with a HA specific monoclonal antibody. Proteinsprecipitated are analyzed on 15% SDS-PAGE gels.

EXAMPLE 6 Expression of Recombinant GPR2 in COS Cells

[0151] The expression of plasmid, GPR2 HA is derived from a vectorpcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E. coli replication origin, 4) CMVpromoter followed by a polylinker region, a SV40 intron andpolyadenylation site. A DNA fragment encoding the entire GPR2 precursorand a HA tag fused in frame to its 3′ end is cloned into the polylinkerregion of the vector, therefore, the recombinant protein expression isdirected under the CMV promoter. The HA tag correspond to an epitopederived from the influenza hemagglutinin protein as previously described(I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R.Lerner, 1984, Cell 37, 767). The infusion of HA tag to the targetprotein allows easy detection of the recombinant protein with anantibody that recognizes the HA epitope.

[0152] The plasmid construction strategy is described as follows:

[0153] The DNA sequence encoding for GPR2, ATCC # 75983, is constructedby PCR using two primers: the 5′ primer 5′GTCCAAGCTTGCCACCATGGTTGGTGGCACCTGG 3′ (SEQ ID No. 19) contains anHindIII site followed by 18 nucleotides of GPR2 coding sequence startingfrom the initiation codon; the 3′ sequence 5′CTAGCTCGAGTCAAGCGTAGTCTGGGACGTCGTATGGGTAGCAGTG GATCCCCCGTGC 3′ (SEQ IDNo. 20) contains complementary sequences to an XhoI site, translationstop codon, HA tag and the last 15 nucleotides of the GPR2 codingsequence (not including the stop codon). Therefore, the PCR productcontains a HindIII site, GPR2 coding sequence followed by HA tag fusedin frame, a translation termination stop codon next to the HA tag, andan XhoI site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp,are digested with HindIII and XhoI restriction enzymes and ligated. Theligation mixture is transformed into E. coli strain SURE (StratageneCloning Systems, La Jolla, Calif.) the transformed culture is plated onampicillin media plates and resistant colonies are selected. Plasmid DNAis isolated from transformants and examined by restriction analysis forthe presence of the correct fragment. For expression of the recombinantGPR2, COS cells are transfected with the expression vector byDEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). Theexpression of the GPR2 HA protein is detected by radiolabelling andimmunoprecipitation method (E. Harlow, D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelledfor 8 hours with ³⁵S-cysteine two days post transfection. Culture mediaare then collected and cells are lysed with detergent (RIPA buffer (150mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5).(Wilson, I. et al., Id. 37: 767 (1984)). Both cell lysate and culturemedia are precipitated with a HA specific monoclonal antibody. Proteinsprecipitated are analyzed on 15% SDS-PAGE gels.

EXAMPLE 7 Expression of Recombinant GPR3 in COS Cells

[0154] The expression of plasmid, GPR3 HA is derived from a vectorpcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E. coli replication origin, 4) CMVpromoter followed by a polylinker region, a SV40 intron andpolyadenylation site. A DNA fragment encoding the entire GPR3 precursorand a HA tag fused in frame to its 3′ end is cloned into the polylinkerregion of the vector, therefore, the recombinant protein expression isdirected under the CMV promoter. The HA tag correspond to an epitopederived from the influenza hemagglutinin protein as previously described(I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R.Lerner, 1984, Cell 37, 767). The infusion of HA tag to the targetprotein allows easy detection of the recombinant protein with anantibody that recognizes the HA epitope.

[0155] The plasmid construction strategy is described as follows:

[0156] The DNA sequence encoding for GPR3, ATCC # 75976, is constructedby PCR using two primers: the 5′ primer 5′GTCCAAGCTTGCCACCATGAACACCACAGTAATG 3′ (SEQ ID No. 21) contains a HindIIIsite followed by 18 nucleotides of GPR3 coding sequence starting fromthe initiation codon; the 3′ sequence 5′CTAGCTCGAGTCAAGCGTAGTCTGGGACGTCGTATGGGTAGCAAGG GATCCATACAAATGT 3′ (SEQID No. 22) contains complementary sequences to an XhoI site, translationstop codon, HA tag and the last 18 nucleotides of the GPR3 codingsequence (not including the stop codon). Therefore, the PCR productcontains a HindIII site, GPR3 coding sequence followed by HA tag fusedin frame, a translation termination stop codon next to the HA tag, andan XhoI site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp,are digested with HindIII and XhoI restriction enzymes and ligated. Theligation mixture is transformed into E. coli strain SURE (StratageneCloning Systems, La Jolla, Calif.) the transformed culture is plated onampicillin media plates and resistant colonies are selected. Plasmid DNAis isolated from transformants and examined by restriction analysis forthe presence of the correct fragment. For expression of the recombinantGPR3, COS cells are transfected with the expression vector byDEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). Theexpression of the GPR3 HA protein is detected by radiolabelling andimmunoprecipitation method (E. Harlow, D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelledfor 8 hours with ³⁵S-cysteine two days post transfection. Culture mediaare then collected and cells are lysed with detergent (RIPA buffer (150mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5)(Wilson, I. et al., Id. 37: 767 (1984)). Both cell lysate and culturemedia are precipitated with a HA specific monoclonal antibody. Proteinsprecipitated are analyzed on 15% SDS-PAGE gels.

EXAMPLE 8 Expression of Recombinant GPR4 in COS Cells

[0157] The expression of plasmid, GPR4 HA is derived from a vectorpcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E. coli replication origin, 4) CMVpromoter followed by a polylinker region, a SV40 intron andpolyadenylation site. A DNA fragment encoding the entire GPR4 precursorand a HA tag fused in frame to its 3′ end is cloned into the polylinkerregion of the vector, therefore, the recombinant protein expression isdirected under the CMV promoter. The HA tag correspond to an epitopederived from the influenza hemagglutinin protein as previously described(I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R.Lerner, 1984, Cell 37, 767). The infusion of HA tag to the targetprotein allows easy detection of the recombinant protein with anantibody that recognizes the HA epitope.

[0158] The plasmid construction strategy is described as follows:

[0159] The DNA sequence encoding for GPR4, ATCC # 75979, is constructedby PCR using two primers: the 5′ primer 5′GTCCAAGCTTGCCACCATGGTAAGCGTTAACAGC 3′ (SEQ ID No. 23) contains a HindIIIsite followed by 18 nucleotides of GPR4 coding sequence starting fromthe initiation codon; the 3′ sequence 5′CTAGCTCGAGTCAAGCGTAGTCTGGGACGTCGTATGGGTAGCAGG CAGCAGATTCATTGTC 3′ (SEQID No. 24) contains complementary sequences to an XhoI site, translationstop codon, HA tag and the last 18 nucleotides of the GPR4 codingsequence (not including the stop codon). Therefore, the PCR productcontains a HindIII site, GPR4 coding sequence followed by HA tag fusedin frame, a translation termination stop codon next to the HA tag, andan XhoI site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp,are digested with Hind III and XhoI restriction enzymes and ligated. Theligation mixture is transformed into E. coli strain SURE (StratageneCloning Systems, La Jolla, Calif.) the transformed culture is plated onampicillin media plates and resistant colonies are selected. Plasmid DNAis isolated from transformants and examined by restriction analysis forthe presence of the correct fragment. For expression of the recombinantGPR4, COS cells are transfected with the expression vector byDEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). Theexpression of the GPR4 HA protein is detected by radiolabelling andimmunoprecipitation method (E. Harlow, D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelledfor 8 hours with ³⁵S-cysteine two days post transfection. Culture mediaare then collected and cells are lysed with detergent (RIPA buffer (150mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5)(Wilson, I. et al., Id. 37: 767 (1984)). Both cell lysate and culturemedia are precipitated with a HA specific monoclonal antibody. Proteinsprecipitated are analyzed on 15% SDS-PAGE gels.

EXAMPLE 9 Cloning and Expression of GPR1 Using the BaculovirusExpression System

[0160] The DNA sequence encoding the full length GPR1 protein, ATCC #75981, is amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ sequences of the gene:

[0161] The 5′ primer has the sequence 5′ CGGGATCCCTCCATGAG TAGTGAAATGGTG3′ (SEQ ID No. 25) and contains a BamHI restriction enzyme site (inbold) followed by 4 nucleotides resembling an efficient signal for theinitiation of translation in eukaryotic cells (Kozak, M., J. Mol. Biol.,196: 947-950 (1987) which is just behind the first 18 nucleotides of theGPR1 gene (the initiation codon for translation “ATG” is underlined).

[0162] The 3′ primer has the sequence 5′ CGGGATCCCGCT CAGGGTTGTAAATCAGG3′ (SEQ ID No. 26) and contains the cleavage site for the BamHIrestriction endonuclease and 18 nucleotides complementary to the 3′non-translated sequence of the GPR1 gene. The amplified sequences areisolated from a 1% agarose gel using a commercially available kit(“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment is thendigested with the endonuclease BamHI and then purified again on a 1%agarose gel. This fragment is designated F2.

[0163] The vector pRG1 (modification of pVL941 vector, discussed below)is used for the expression of the GPR1 protein using the baculovirusexpression system (for review see: Summers, M. D. and Smith, G. E. 1987,A manual of methods for baculovirus vectors and insect cell cultureprocedures, Texas Agricultural Experimental Station Bulletin No. 1555).This expression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonuclease BamHI. Thepolyadenylation site of the simian virus (SV)40 is used for efficientpolyadenylation. For an easy selection of recombinant virus thebeta-galactosidase gene from E. coli is inserted in the same orientationas the polyhedrin promoter followed by the polyadenylation signal of thepolyhedrin gene. The polyhedrin sequences are flanked at both sides byviral sequences for the cell-mediated homologous recombination ofcotransfected wild-type viral DNA. Many other baculovirus vectors couldbe used in place of pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summers, M. D., Virology, 170: 31-39).

[0164] The plasmid is digested with the restriction enzymes BamHI andthen dephosphorylated using calf intestinal phosphatase by proceduresknown in the art. The DNA is then isolated from a 1% agarose gel usingthe commercially available kit (“Geneclean” BIO 101 Inc., La Jolla,Calif.). This vector DNA is designated V2.

[0165] Fragment F2 and the dephosphorylated plasmid V2 are ligated withT4 DNA ligase. E. coli HB101 cells are then transformed and bacteriaidentified that contained the plasmid (pBacGPR1) with the GPR1 geneusing the enzymes BamHI. The sequence of the cloned fragment isconfirmed by DNA sequencing.

[0166] 5 μg of the plasmid pBacGPR1 is cotransfected with 1.0 μg of acommercially available linearized baculovirus (“BaculoGold™ baculovirusDNA”, Pharmingen, San Diego, Calif..) using the lipofection method(Felgner et al. Proc. Natl. Acad. Sci. USA, 84: 7413-7417 (1987)).

[0167] 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBacGPR1are mixed in a sterile well of a microtiter plate containing 50 μl ofserum free Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture is added dropwise to the Sf9 insect cells (ATCC CRL 1711) seededin a 35 mm tissue culture plate with 1 ml Grace's medium without serum.The plate is rocked back and forth to mix the newly added solution. Theplate is then incubated for 5 hours at 27° C. After 5 hours thetransfection solution is removed from the plate and 1 ml of Grace'sinsect medium supplemented with 10% fetal calf serum is added. The plateis put back into an incubator and cultivation continued at 27° C. forfour days.

[0168] After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

[0169] Four days after the serial dilution, the virus are added to thecells, blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses is then resuspendedin an Eppendorf tube containing 200 μl of Grace's medium. The agar isremoved by a brief centrifugation and the supernatant containing therecombinant baculovirus is used to infect Sf9 cells seeded in 35 mmdishes. Four days later the supernatants of these culture dishes areharvested and then stored at 4° C.

[0170] Sf9 cells are grown in Grace's medium supplemented with 10 %heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-GPR1 at a multiplicity of infection (MOI) of 2. Six hourslater the medium is removed and replaced with SF900 II medium minusmethionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hourslater 5 μCi of ³⁵S-methionine and 5 μCi ³⁵S cysteine (Amersham) areadded. The cells are further incubated for 16 hours before they areharvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

EXAMPLE 5 Expression Via Gene Therapy

[0171] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

[0172] pMV-7 (Kirschmeier, P. T. et al, DNA, 7: 219-25 (1988) flanked bythe long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0173] The cDNA encoding a polypeptide of the present invention isamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively. The 5′ primer containing an EcoRI site and the3′ primer $further includes a HindIII site. Equal quantities of theMoloney murine sarcoma virus linear backbone and the amplified $EcoRIand HindIII fragment are added together, in the presence of T4 DNAligase. The resulting mixture is maintained under conditions appropriatefor ligation of the two fragments. The ligation mixture is used totransform bacteria HB101, which are then plated onto agar-containingkanamycin for the purpose of confirming that the vector had the gene ofinterest properly inserted.

[0174] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells are transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[0175] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his.

[0176] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product.

[0177] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1 30 1713 base pairs nucleic acid both both cDNA CDS 116..1003 1GGCACGAGGT CATTCAACAT TTATTCAACC AAAAATACTA AGTCAGCTCT ATACAAACTA 60ATGGAAGGAT ACAGCTATGC AAATATAGAA CACTAAAGTG TTACATGACA GATGT ATG 118 Met1 AGT AGT GAA ATG GTG AAA AAT CAG ACA ATG GTC ACA GAG TTC CTC CTA 166Ser Ser Glu Met Val Lys Asn Gln Thr Met Val Thr Glu Phe Leu Leu 5 10 15CTG GGA TTT CTC CTG GGC CCA AGG ATT CAG ATG CTC CTC TTT GGG CTC 214 LeuGly Phe Leu Leu Gly Pro Arg Ile Gln Met Leu Leu Phe Gly Leu 20 25 30 TTCTCC CTG TTC TAT GTC TTC ACC CTG CTG GGG AAT GGG ACC ATC CTG 262 Phe SerLeu Phe Tyr Val Phe Thr Leu Leu Gly Asn Gly Thr Ile Leu 35 40 45 GGG CTCATC TCA CTG GAC TCC AGA CTC CAC ACC CCC ATG TAC TTC TTC 310 Gly Leu IleSer Leu Asp Ser Arg Leu His Thr Pro Met Tyr Phe Phe 50 55 60 65 CTC TCACAC CTG GCC GTC GTC AAC ATC GCC TAT GCC TGC AAC ACA GTG 358 Leu Ser HisLeu Ala Val Val Asn Ile Ala Tyr Ala Cys Asn Thr Val 70 75 80 CCC CAG ATGCTG GTG AAC CTC CTG CAT CCA GCC AAG CCC ATC TCC TTT 406 Pro Gln Met LeuVal Asn Leu Leu His Pro Ala Lys Pro Ile Ser Phe 85 90 95 GCT GGT TGC ATGACA CTA GAC TTT CTC TTT TTG AGT TTT GCA CAT ACT 454 Ala Gly Cys Met ThrLeu Asp Phe Leu Phe Leu Ser Phe Ala His Thr 100 105 110 GAA TGC CTC CTGTTG GTG CTG ATG TCC TAC GAT CGG TAC GTG GCC ATC 502 Glu Cys Leu Leu LeuVal Leu Met Ser Tyr Asp Arg Tyr Val Ala Ile 115 120 125 TGC CAC CCT CTCCGA TAT TTC ATC ATC ATG ACC TGG AAA GTC TGC ATC 550 Cys His Pro Leu ArgTyr Phe Ile Ile Met Thr Trp Lys Val Cys Ile 130 135 140 145 ACT CTG GGCATC ACT TCC TGG ACA TGT GGC TCC CTC CTG GCT ATG GTC 598 Thr Leu Gly IleThr Ser Trp Thr Cys Gly Ser Leu Leu Ala Met Val 150 155 160 CAT GTG AGCCTC ATC CTA AGA CTG CCC TTT TGT GGG CCT CGT GAA ATC 646 His Val Ser LeuIle Leu Arg Leu Pro Phe Cys Gly Pro Arg Glu Ile 165 170 175 AAC CAC TTCTTC TGT GAA ATC CTG TCT GTC CTC AGG CTG GCC TGT GCT 694 Asn His Phe PheCys Glu Ile Leu Ser Val Leu Arg Leu Ala Cys Ala 180 185 190 GAT ACC TGGCTC AAC CAG GTG GTC ATC TTT GAA GCC TGC ATG TTC ATC 742 Asp Thr Trp LeuAsn Gln Val Val Ile Phe Glu Ala Cys Met Phe Ile 195 200 205 CTG GTG GGACCA CTC TGC CTG GTG CTG GTC TCC TAC TCA CAC ATC CTG 790 Leu Val Gly ProLeu Cys Leu Val Leu Val Ser Tyr Ser His Ile Leu 210 215 220 225 GGG GGCATC CTG AGG ATC CAG TCT GGG GAG GGC CGC AGA AAG GCC TTC 838 Gly Gly IleLeu Arg Ile Gln Ser Gly Glu Gly Arg Arg Lys Ala Phe 230 235 240 TCC ACCTGC TCC TCC CAC CTC TGC GTA GTG GGA CTC TTC TTT GGS AGC 886 Ser Thr CysSer Ser His Leu Cys Val Val Gly Leu Phe Phe Gly Ser 245 250 255 GCC ATCGTC ATG TAC ATG GCC CCT AAG TCC CGC CAT CCT GAG GAG CAG 934 Ala Ile ValMet Tyr Met Ala Pro Lys Ser Arg His Pro Glu Glu Gln 260 265 270 CAG AAGGTC CTT TTT CTT ATT TTA CAG TTC CTT TCA ACC CCG ATG CTT 982 Gln Lys ValLeu Phe Leu Ile Leu Gln Phe Leu Ser Thr Pro Met Leu 275 280 285 AAA CCCCCT GAT TTA CAA CCC TGA GGAATGTAGA GGGTCAAGGG TGCCCTCCGA 1036 Lys ProPro Asp Leu Gln Pro 290 295 GGAGACCACT GTGCAARGRA AGTCATTCCT AAGGGGTGTGACATTTGAAC TGCCAGCCCC 1096 AGTTGCCCCG TGGACTCCTG ATGCCCAATT ATTGCCTCAACCCAGAAAAG TTTACTCCCC 1156 TTTAACTGTG CTTTACTGAC AGAAGGGCAA GCCTTCTCCCGTTTTTTGCA GATAAAATTT 1216 TAGATGTGTT GCAATCATTG GGTTTCTAGG AGATGTGGTTTTATCAGACA ATTTTTTCTT 1276 TTATTTCACA ATTACTTTAA TATCTGTAAA ATAAAGAATTATTTTAAATC ATTTTCCCAG 1336 TCCCAAAAGT TAAATACAGG CCACTTACTT CTTTAACCAAATGATATAGT TTGGCTCTGT 1396 GTCCCCACCC AAATCTCATG TCAAATTGTA ATCCCCGCATGTCAGCGGAG GGACCTGGTG 1456 GGAGGTGATT GGATCATGGG GAGGGATTTC CCCCTTGCTGTTCTGTTGAT AGTGAACGAG 1516 TTCTCACGAA ATCTGATGGT TTAAAAGTGC AGCACTTCTCCCTTTGCTCT CTCTCTCCTG 1576 CTGTGCCATG GTAAGACGTG CCTTGCTTCC CCTGGTGCTTCCGCCATGAT TGTACCTTTC 1636 CTGAGGCCTC TCCAGCCATG TGGAACTGTG AGCCAATTAAACTTCTTTTC TTTAGAAAAA 1696 AAAAAAAAAA AAAAAAA 1713 296 amino acids aminoacid linear protein 2 Met Ser Ser Glu Met Val Lys Asn Gln Thr Met ValThr Glu Phe Leu 1 5 10 15 Leu Leu Gly Phe Leu Leu Gly Pro Arg Ile GlnMet Leu Leu Phe Gly 20 25 30 Leu Phe Ser Leu Phe Tyr Val Phe Thr Leu LeuGly Asn Gly Thr Ile 35 40 45 Leu Gly Leu Ile Ser Leu Asp Ser Arg Leu HisThr Pro Met Tyr Phe 50 55 60 Phe Leu Ser His Leu Ala Val Val Asn Ile AlaTyr Ala Cys Asn Thr 65 70 75 80 Val Pro Gln Met Leu Val Asn Leu Leu HisPro Ala Lys Pro Ile Ser 85 90 95 Phe Ala Gly Cys Met Thr Leu Asp Phe LeuPhe Leu Ser Phe Ala His 100 105 110 Thr Glu Cys Leu Leu Leu Val Leu MetSer Tyr Asp Arg Tyr Val Ala 115 120 125 Ile Cys His Pro Leu Arg Tyr PheIle Ile Met Thr Trp Lys Val Cys 130 135 140 Ile Thr Leu Gly Ile Thr SerTrp Thr Cys Gly Ser Leu Leu Ala Met 145 150 155 160 Val His Val Ser LeuIle Leu Arg Leu Pro Phe Cys Gly Pro Arg Glu 165 170 175 Ile Asn His PhePhe Cys Glu Ile Leu Ser Val Leu Arg Leu Ala Cys 180 185 190 Ala Asp ThrTrp Leu Asn Gln Val Val Ile Phe Glu Ala Cys Met Phe 195 200 205 Ile LeuVal Gly Pro Leu Cys Leu Val Leu Val Ser Tyr Ser His Ile 210 215 220 LeuGly Gly Ile Leu Arg Ile Gln Ser Gly Glu Gly Arg Arg Lys Ala 225 230 235240 Phe Ser Thr Cys Ser Ser His Leu Cys Val Val Gly Leu Phe Phe Gly 245250 255 Ser Ala Ile Val Met Tyr Met Ala Pro Lys Ser Arg His Pro Glu Glu260 265 270 Gln Gln Lys Val Leu Phe Leu Ile Leu Gln Phe Leu Ser Thr ProMet 275 280 285 Leu Lys Pro Pro Asp Leu Gln Pro 290 295 2185 base pairsnucleic acid both both cDNA CDS 884..2062 3 TCACTATAGG GCGAATTGGGTACGGGCCCC CCCTCGAGGT CGACGGTATC GATAAGCTTG 60 ATATCGAATT CGGCACGAGCCGGGCTCGGA GAGGTGACGG AACCGGGGCT GGTAGCATAG 120 TTTGATTTGA TGATGGAGCCAACACAGGGG TTGGAGCTGG TACCGGTGAA GCTGAGGCTA 180 AAAAGGTTCC TGGAGTAGACGATGGAGCCA TAACTGGAAC CGGAGTCTGT GAATGAAGCC 240 AGGACAGGAG CAGCACCTGGCGATGGTGCC AGGACCGGAA GAGGAGCCAG AGGAGGAGCT 300 GGAGAAGGAG CCAGAATTGCTGTCTGTGGA GCCGCCATAG GAGCCAGAGG GGTGGCTAGA 360 GCCTGAGAAT GCAGAAGATGCTGGAGCCAG AAGGGAAGCC TGAGCTGGAG CTGGATTTGG 420 TGCTGACGGA AAAGGACTGGCCAGAGCCGA AGCTGGCACC AGGGACAGGT GAGCATTCTG 480 GGGCCACGGT TGAGTTCAACCCACTGACTT CAGGTGAAGG ACTGTGGACC AGCTTGAGAA 540 GAGGCCTCAC CAGAGTGGGTGTGGGGCATG GGGGCTCGAG CAGTACCCAG AGTAGGTGTG 600 GGTAGCCCGG CCAGGGGTTAACGTGGGGCG TGGATTCAAC ACAGCTTGGA AGCCCAGAGC 660 TCGGAGGCCC GGGTGCTTGGGCCAATTGAG GAACAGGAGT CAGTCCATCC CGAGGGGGTT 720 GTCTCACTAC AATCTTCACACGCCTTTATT ATTCACCATG GTTGGTGGCA CCTGGTTAGC 780 AGCAAGCGGA AGGCTGAGGCCAGTAGGGGC AGGGGTGTTA CTGGGGGTCG AAGAAGCCAG 840 CACAGAGACA GGGGTAGGGCCAGGGGTCGG GGCCACGGCC TGG ATG AGG CCC ACA 895 Met Arg Pro Thr 1 TGG GCAGGC TGG CTG ATG AGA TGG TGC TGC CCC CCT GCT GAC ACG AGG 943 Trp Ala GlyTrp Leu Met Arg Trp Cys Cys Pro Pro Ala Asp Thr Arg 5 10 15 20 TGC ACCACA TTC CTT TGC AGC GGG CGG GCT GCC CCA CAG CAA GCT GGC 991 Cys Thr ThrPhe Leu Cys Ser Gly Arg Ala Ala Pro Gln Gln Ala Gly 25 30 35 GCA CCT GGGCAC CAT CCA AAA TAC AGC TTG TTT CCC TGG ATT TGG AAG 1039 Ala Pro Gly HisHis Pro Lys Tyr Ser Leu Phe Pro Trp Ile Trp Lys 40 45 50 GTG AGA GGT TTGCTT CCC CCT CCA TTA ACC ACT GAC GTT GTG CCA GTG 1087 Val Arg Gly Leu LeuPro Pro Pro Leu Thr Thr Asp Val Val Pro Val 55 60 65 AGA CTA ACT CTC CGCGCC AAT CTG TCC GCG GCT GAC CTC CTT CGC GGG 1135 Arg Leu Thr Leu Arg AlaAsn Leu Ser Ala Ala Asp Leu Leu Arg Gly 70 75 80 CGT GGC CTA CCT CTT CCTCAT GTT CCA CAC TGT CCC CGC ACA GCC CGA 1183 Arg Gly Leu Pro Leu Pro HisVal Pro His Cys Pro Arg Thr Ala Arg 85 90 95 100 CTT TCA CTT GAG GGC TGGTTC CTG CGG CAG GGC TTG CTG GAC ACA AAC 1231 Leu Ser Leu Glu Gly Trp PheLeu Arg Gln Gly Leu Leu Asp Thr Asn 105 110 115 CTC ACT GCG TCG GTG GCCACA CTG CTG GCC ATC GCC GTG GAG CGG CAC 1279 Leu Thr Ala Ser Val Ala ThrLeu Leu Ala Ile Ala Val Glu Arg His 120 125 130 CGC AGT GTG ATG GCC GTGCAG CTG CAC AGC CGC CTG CCC CGT GGC CGC 1327 Arg Ser Val Met Ala Val GlnLeu His Ser Arg Leu Pro Arg Gly Arg 135 140 145 GTG GTC ATG CTC ATT GTGGGC GTG TGG GTG GCT GCC CTG GGC CTG GGG 1375 Val Val Met Leu Ile Val GlyVal Trp Val Ala Ala Leu Gly Leu Gly 150 155 160 CTG CTG CCT GCC CAC TCCTGG CAC TGC CTC TGT GCC CTG GAC CGC TCC 1423 Leu Leu Pro Ala His Ser TrpHis Cys Leu Cys Ala Leu Asp Arg Ser 165 170 175 180 TCA CGC ATG GCA CCCCTG CTC AGC CGC TCC TAT TTG GCC GTC TGG GCT 1471 Ser Arg Met Ala Pro LeuLeu Ser Arg Ser Tyr Leu Ala Val Trp Ala 185 190 195 CTG TCG AGC CTG CTTGTC TTC CTG CTC ATG GTG GCT GTG TAC ACC CGC 1519 Leu Ser Ser Leu Leu ValPhe Leu Leu Met Val Ala Val Tyr Thr Arg 200 205 210 ATT TTC TTC TAC GTGCGG CGG CGA GTG CAG CGC ATG GCA GAG CAT GTC 1567 Ile Phe Phe Tyr Val ArgArg Arg Val Gln Arg Met Ala Glu His Val 215 220 225 AGC TGC CAC CCC CGCTAC CGA GAG ACC ACG CTC AGC CTG GTC AAG ACT 1615 Ser Cys His Pro Arg TyrArg Glu Thr Thr Leu Ser Leu Val Lys Thr 230 235 240 GTT GTC ATC ATC CTGGGG GCG TTC GTG GTC TGC TGG ACA CCA GGC CAG 1663 Val Val Ile Ile Leu GlyAla Phe Val Val Cys Trp Thr Pro Gly Gln 245 250 255 260 GTG GTA CTG CTCCTG GAT GGT TTA GGC TGT GAG TCC TGC AAT GTC CTG 1711 Val Val Leu Leu LeuAsp Gly Leu Gly Cys Glu Ser Cys Asn Val Leu 265 270 275 GCG TTA GAA AAGTAC TTC CTA CTG TTG GCC GAG CCA ACC TCA CTG GTC 1759 Ala Leu Glu Lys TyrPhe Leu Leu Leu Ala Glu Pro Thr Ser Leu Val 280 285 290 AAT GCT GCT GTGTAC TCT TGC CGA GAT GCT GAG ATG CGC CGC ACC TTC 1807 Asn Ala Ala Val TyrSer Cys Arg Asp Ala Glu Met Arg Arg Thr Phe 295 300 305 CGC CGC CTT CTCCTG CTG CGC GTG CCT CCG CCA GTC CAC CCG CGA GTC 1855 Arg Arg Leu Leu LeuLeu Arg Val Pro Pro Pro Val His Pro Arg Val 310 315 320 TGT CCA CTA TACATC CTC TGC CCA GGG AGG TGC CAG CAC TCG CAT CAT 1903 Cys Pro Leu Tyr IleLeu Cys Pro Gly Arg Cys Gln His Ser His His 325 330 335 340 GCT TCC CGAGAA CGG CCA CCC ACT GAT GGA CTC CAC CCT TTA GCT ACC 1951 Ala Ser Arg GluArg Pro Pro Thr Asp Gly Leu His Pro Leu Ala Thr 345 350 355 TTG AAC TACAGC GGT ACG CGG CAA GCA ACA AAT CCA CAG CCC CTG ATG 1999 Leu Asn Tyr SerGly Thr Arg Gln Ala Thr Asn Pro Gln Pro Leu Met 360 365 370 ACT TGT GGGTGC TCC TGG CTC AAC CCA ACC TCG TGC CGA ATT CCT GCA 2047 Thr Cys Gly CysSer Trp Leu Asn Pro Thr Ser Cys Arg Ile Pro Ala 375 380 385 GCC CGG GGGATC CAC TAG TTCTAGAGCG GCGCCACCGC GGTGGAGCTC 2095 Ala Arg Gly Ile His390 CAGCTTTTGT TCCCTTTAGT GAGGGTTAAT TTCGAGCTTG GCGTAATCAT GGTCATAGCT2155 GTTTCCTGTG TGAAATTGTT ATCCGCTCAC 2185 393 amino acids amino acidlinear protein 4 Met Arg Pro Thr Trp Ala Gly Trp Leu Met Arg Trp Cys CysPro Pro 1 5 10 15 Ala Asp Thr Arg Cys Thr Thr Phe Leu Cys Ser Gly ArgAla Ala Pro 20 25 30 Gln Gln Ala Gly Ala Pro Gly His His Pro Lys Tyr SerLeu Phe Pro 35 40 45 Trp Ile Trp Lys Val Arg Gly Leu Leu Pro Pro Pro LeuThr Thr Asp 50 55 60 Val Val Pro Val Arg Leu Thr Leu Arg Ala Asn Leu SerAla Ala Asp 65 70 75 80 Leu Leu Arg Gly Arg Gly Leu Pro Leu Pro His ValPro His Cys Pro 85 90 95 Arg Thr Ala Arg Leu Ser Leu Glu Gly Trp Phe LeuArg Gln Gly Leu 100 105 110 Leu Asp Thr Asn Leu Thr Ala Ser Val Ala ThrLeu Leu Ala Ile Ala 115 120 125 Val Glu Arg His Arg Ser Val Met Ala ValGln Leu His Ser Arg Leu 130 135 140 Pro Arg Gly Arg Val Val Met Leu IleVal Gly Val Trp Val Ala Ala 145 150 155 160 Leu Gly Leu Gly Leu Leu ProAla His Ser Trp His Cys Leu Cys Ala 165 170 175 Leu Asp Arg Ser Ser ArgMet Ala Pro Leu Leu Ser Arg Ser Tyr Leu 180 185 190 Ala Val Trp Ala LeuSer Ser Leu Leu Val Phe Leu Leu Met Val Ala 195 200 205 Val Tyr Thr ArgIle Phe Phe Tyr Val Arg Arg Arg Val Gln Arg Met 210 215 220 Ala Glu HisVal Ser Cys His Pro Arg Tyr Arg Glu Thr Thr Leu Ser 225 230 235 240 LeuVal Lys Thr Val Val Ile Ile Leu Gly Ala Phe Val Val Cys Trp 245 250 255Thr Pro Gly Gln Val Val Leu Leu Leu Asp Gly Leu Gly Cys Glu Ser 260 265270 Cys Asn Val Leu Ala Leu Glu Lys Tyr Phe Leu Leu Leu Ala Glu Pro 275280 285 Thr Ser Leu Val Asn Ala Ala Val Tyr Ser Cys Arg Asp Ala Glu Met290 295 300 Arg Arg Thr Phe Arg Arg Leu Leu Leu Leu Arg Val Pro Pro ProVal 305 310 315 320 His Pro Arg Val Cys Pro Leu Tyr Ile Leu Cys Pro GlyArg Cys Gln 325 330 335 His Ser His His Ala Ser Arg Glu Arg Pro Pro ThrAsp Gly Leu His 340 345 350 Pro Leu Ala Thr Leu Asn Tyr Ser Gly Thr ArgGln Ala Thr Asn Pro 355 360 365 Gln Pro Leu Met Thr Cys Gly Cys Ser TrpLeu Asn Pro Thr Ser Cys 370 375 380 Arg Ile Pro Ala Ala Arg Gly Ile His385 390 1474 base pairs nucleic acid both both cDNA CDS 62..940 5CGGCACGAGC ATAAGAAGAC AGAGAGAACT GAGTATCCTC CCAAAGGTGA CACTGGAAGC 60 AATG AAC ACC ACA GTA ATG CAA GGC TTC AAC AGA TCT AAG CGG TGC 106 Met AsnThr Thr Val Met Gln Gly Phe Asn Arg Ser Lys Arg Cys 1 5 10 15 CCC AAAGAC ACT CGG ATA GTA CAG CTG GTA TTC CCA GCC CTC TAC ACA 154 Pro Lys AspThr Arg Ile Val Gln Leu Val Phe Pro Ala Leu Tyr Thr 20 25 30 GTG GTT TTCTTG ACC GGA ATC CTG CTG AAT ACT TTG GCT CTG TGG GTG 202 Val Val Phe LeuThr Gly Ile Leu Leu Asn Thr Leu Ala Leu Trp Val 35 40 45 TTT GTT CAC ATCCCC AGC TCC TCC ACC TTC ATC ATC TAC CTC AAA AAC 250 Phe Val His Ile ProSer Ser Ser Thr Phe Ile Ile Tyr Leu Lys Asn 50 55 60 ACT TTG GTG GCC GACTTG ATA ATG ACA CTC ATG CTT CCT TTC AAA ATC 298 Thr Leu Val Ala Asp LeuIle Met Thr Leu Met Leu Pro Phe Lys Ile 65 70 75 CTC TCT GAC TCA CAC CTGGCA CCC TGG CAG CTC AGA GCT TTT GTG TGT 346 Leu Ser Asp Ser His Leu AlaPro Trp Gln Leu Arg Ala Phe Val Cys 80 85 90 95 CGT TTT TCT TCG GTG ATATTT TAT GAG ACC ATG TAT GTG GGC ATC GTG 394 Arg Phe Ser Ser Val Ile PheTyr Glu Thr Met Tyr Val Gly Ile Val 100 105 110 CTG TTA GGG CTC ATA GCCTTT GAC AGA TTC CTC AAG ATC ATC AGA CCT 442 Leu Leu Gly Leu Ile Ala PheAsp Arg Phe Leu Lys Ile Ile Arg Pro 115 120 125 TTG AGA AAT ATT TTT CTAAAA AAA CCT GTT TGG GGA AAA ACG GTC TCA 490 Leu Arg Asn Ile Phe Leu LysLys Pro Val Trp Gly Lys Thr Val Ser 130 135 140 ATC TTC ATC TGG TTC TTTTGG TTC TTC ATC TCC CTG CCA AAT ATG ATC 538 Ile Phe Ile Trp Phe Phe TrpPhe Phe Ile Ser Leu Pro Asn Met Ile 145 150 155 TTG AGC AAC AAG GAA GCAACA CCA TCG TCT GTG AAA AAG TGT GCT TCC 586 Leu Ser Asn Lys Glu Ala ThrPro Ser Ser Val Lys Lys Cys Ala Ser 160 165 170 175 TTA AAG GGG CCT CTGGGG CTG AAA TGG CAT CAA ATG GTA AAT AAC ATA 634 Leu Lys Gly Pro Leu GlyLeu Lys Trp His Gln Met Val Asn Asn Ile 180 185 190 TGC CAG TTT ATT TTCTGG ACT GTT TTT ATC CTA ATG CTT GTG TTT TAT 682 Cys Gln Phe Ile Phe TrpThr Val Phe Ile Leu Met Leu Val Phe Tyr 195 200 205 GTG GTT ATT GCA AAAAAG TAT ATG ATT CTT ATA GAA AGT CCA AAA GTA 730 Val Val Ile Ala Lys LysTyr Met Ile Leu Ile Glu Ser Pro Lys Val 210 215 220 AGG ACA GAA AAA ACAACA AAA AGC TGG AAG GCA AAG TAT TTG TTG TCG 778 Arg Thr Glu Lys Thr ThrLys Ser Trp Lys Ala Lys Tyr Leu Leu Ser 225 230 235 TGG CTG TCT TCT TTGTGT GTT TTG CTC CAT TTC ATT TCG CCA GAG TTC 826 Trp Leu Ser Ser Leu CysVal Leu Leu His Phe Ile Ser Pro Glu Phe 240 245 250 255 CAT ATA CTC ACAGTC AAA CCA ACA ATA AGA CTG ACT GTA GAC TGC AAA 874 His Ile Leu Thr ValLys Pro Thr Ile Arg Leu Thr Val Asp Cys Lys 260 265 270 ATC AAC TGT TTATTG CTA AAG AAA CAA CTC TCT TTT TGG CAG CAA CTA 922 Ile Asn Cys Leu LeuLeu Lys Lys Gln Leu Ser Phe Trp Gln Gln Leu 275 280 285 ACA TTT GTA TGGATC CCT TAA TATACATATT CTTATGTAAA AAATTCACAG 973 Thr Phe Val Trp Ile Pro290 AAAAGCTACC ATGTATGCAA GGGAGAAAGA CCACAGCATC AAGCCAAGAA AATCATAGCA1033 GTCAGACAGA CAACATAACC TTAGGCTGAC AACTGTACAT AGGGGTAACT TCTATTTATT1093 GATGAGACTT CCGTAGATAA TGTGGAAATC CAATTTAACC AAGAAAAAAA GATTGGGGCA1153 AATGCTCTCT TACATTTTAT TATCCTGGTG TACAGAAAAG ATTATATAAA ATTTAAATCC1213 ACATAGATCT ATTCATAAGC TGAATGAACC ATTACTAAGA GAATGCAACA GGATACAAAT1273 GGCCACTAGA GGTCATTATT TCTTTCTTTC TTTCTTTTTT TTTTTTTAAT TTCAAGAGCA1333 TTTCACTTTA ACATTTTGGA AAAGACTAAG GAGAAACGTA TATCCCTACA AACCTCCCCT1393 CCAAACACCT TCTTACATTC TTTTCCACAA TTCACATAAC ACTACTGCTT TTGTGCCCCT1453 TAAATGTAGA TTTGTTGGCT G 1474 293 amino acids amino acid linearprotein 6 Met Asn Thr Thr Val Met Gln Gly Phe Asn Arg Ser Lys Arg CysPro 1 5 10 15 Lys Asp Thr Arg Ile Val Gln Leu Val Phe Pro Ala Leu TyrThr Val 20 25 30 Val Phe Leu Thr Gly Ile Leu Leu Asn Thr Leu Ala Leu TrpVal Phe 35 40 45 Val His Ile Pro Ser Ser Ser Thr Phe Ile Ile Tyr Leu LysAsn Thr 50 55 60 Leu Val Ala Asp Leu Ile Met Thr Leu Met Leu Pro Phe LysIle Leu 65 70 75 80 Ser Asp Ser His Leu Ala Pro Trp Gln Leu Arg Ala PheVal Cys Arg 85 90 95 Phe Ser Ser Val Ile Phe Tyr Glu Thr Met Tyr Val GlyIle Val Leu 100 105 110 Leu Gly Leu Ile Ala Phe Asp Arg Phe Leu Lys IleIle Arg Pro Leu 115 120 125 Arg Asn Ile Phe Leu Lys Lys Pro Val Trp GlyLys Thr Val Ser Ile 130 135 140 Phe Ile Trp Phe Phe Trp Phe Phe Ile SerLeu Pro Asn Met Ile Leu 145 150 155 160 Ser Asn Lys Glu Ala Thr Pro SerSer Val Lys Lys Cys Ala Ser Leu 165 170 175 Lys Gly Pro Leu Gly Leu LysTrp His Gln Met Val Asn Asn Ile Cys 180 185 190 Gln Phe Ile Phe Trp ThrVal Phe Ile Leu Met Leu Val Phe Tyr Val 195 200 205 Val Ile Ala Lys LysTyr Met Ile Leu Ile Glu Ser Pro Lys Val Arg 210 215 220 Thr Glu Lys ThrThr Lys Ser Trp Lys Ala Lys Tyr Leu Leu Ser Trp 225 230 235 240 Leu SerSer Leu Cys Val Leu Leu His Phe Ile Ser Pro Glu Phe His 245 250 255 IleLeu Thr Val Lys Pro Thr Ile Arg Leu Thr Val Asp Cys Lys Ile 260 265 270Asn Cys Leu Leu Leu Lys Lys Gln Leu Ser Phe Trp Gln Gln Leu Thr 275 280285 Phe Val Trp Ile Pro 290 1301 base pairs nucleic acid both both cDNACDS 161..1192 7 TTTTGGGTAT TTCTGAGAAA AAGGAAATAT TTATAAAACC ATCCAAAGATCCAGATAATT 60 TGCAAATAAA TTGGAGGTTA TAGAGGTTAT AATCTGAATC CCAAAGGAGACTGCAGCTGA 120 TGAAAGTGCT TCCAAACTGA AAATTGGACG TGCCTTTACG ATG GTA AGCGTT AAC 175 Met Val Ser Val Asn 1 5 AGC TCC CAC TGC TTC TAT AAT GAC TCCTTT AAG TAC ACT TTG TAT GGG 223 Ser Ser His Cys Phe Tyr Asn Asp Ser PheLys Tyr Thr Leu Tyr Gly 10 15 20 TGC ATG TTC AGC ATG GTG TTT GTG CTT GGGTTA ATA TCC AAT TGT GTT 271 Cys Met Phe Ser Met Val Phe Val Leu Gly LeuIle Ser Asn Cys Val 25 30 35 GCC ATA TAC ATT TTC ATC TGC GTC CTC AAA GTCCGA AAT GAA ACT ACA 319 Ala Ile Tyr Ile Phe Ile Cys Val Leu Lys Val ArgAsn Glu Thr Thr 40 45 50 ACT TAC ATG ATT AAC TTG GCA ATG TCA GAC TTG CTTTTT GTT TTT ACT 367 Thr Tyr Met Ile Asn Leu Ala Met Ser Asp Leu Leu PheVal Phe Thr 55 60 65 TTA CCC TTC AGG ATT TTT TAC TTC ACA ACA CGG AAT TGGCCA TTT GGA 415 Leu Pro Phe Arg Ile Phe Tyr Phe Thr Thr Arg Asn Trp ProPhe Gly 70 75 80 85 GAT TTA CTT TGT AAG ATT TCT GTG ATG CTG TTT TAT ACCAAC ATG TAC 463 Asp Leu Leu Cys Lys Ile Ser Val Met Leu Phe Tyr Thr AsnMet Tyr 90 95 100 GGA AGC ATT CTG TTC TTA ACC TGT ATT AGT GTA GAT CGATTT CTG GCA 511 Gly Ser Ile Leu Phe Leu Thr Cys Ile Ser Val Asp Arg PheLeu Ala 105 110 115 ATT GTC TAC CCA TTT AAG TCA AAG ACT CTA AGA ACC AAAAGA AAT GCA 559 Ile Val Tyr Pro Phe Lys Ser Lys Thr Leu Arg Thr Lys ArgAsn Ala 120 125 130 AAG ATT GTT TGC ACT GGC GTG TGG TTA ACT GTG ATC GGAGGA AGT GCA 607 Lys Ile Val Cys Thr Gly Val Trp Leu Thr Val Ile Gly GlySer Ala 135 140 145 CCC GCC GTT TTT GTT CAG TCT ACC CAC TCT CAG GGT AACAAT GCC TCA 655 Pro Ala Val Phe Val Gln Ser Thr His Ser Gln Gly Asn AsnAla Ser 150 155 160 165 GAA GCC TGC TTT GAA AAT TTT CCA GAA GCC ACA TGGAAA ACA TAT CTC 703 Glu Ala Cys Phe Glu Asn Phe Pro Glu Ala Thr Trp LysThr Tyr Leu 170 175 180 TCA AGG ATT GTA ATT TTC ATC GAA ATA GTG GGA TTTTTT ATT CCT CTA 751 Ser Arg Ile Val Ile Phe Ile Glu Ile Val Gly Phe PheIle Pro Leu 185 190 195 ATT TTA AAT GTA ACT TGT TCT AGT ATG GTG CTA AAAACT TTA ACC AAA 799 Ile Leu Asn Val Thr Cys Ser Ser Met Val Leu Lys ThrLeu Thr Lys 200 205 210 CCT GTT ACA TTA AGT AGA AGC AAA ATA AAC AAA ACTAAG GTT TTA AAA 847 Pro Val Thr Leu Ser Arg Ser Lys Ile Asn Lys Thr LysVal Leu Lys 215 220 225 ATG ATT TTT GTA CAT TTG ATC ATA TTC TGT TTC TGTTTT GTT CCT TAC 895 Met Ile Phe Val His Leu Ile Ile Phe Cys Phe Cys PheVal Pro Tyr 230 235 240 245 AAT ATC AAT CTT ATT TTA TAT TCT CTT GTG AGAACA CAA ACA TTT GTT 943 Asn Ile Asn Leu Ile Leu Tyr Ser Leu Val Arg ThrGln Thr Phe Val 250 255 260 AAT TGC TCA GTA GTG GCA GCA GTA AGG ACA ATGTAC CCA ATC ACT CTC 991 Asn Cys Ser Val Val Ala Ala Val Arg Thr Met TyrPro Ile Thr Leu 265 270 275 TGT ATT GCT GTT TCC AAC TGT TGT TTT GAC CCTATA GTT TAC TAC TTT 1039 Cys Ile Ala Val Ser Asn Cys Cys Phe Asp Pro IleVal Tyr Tyr Phe 280 285 290 ACA TCG GAC ACA ATT CAG AAT TCA ATA AAA ATGAAA AAC TGG TCT GTC 1087 Thr Ser Asp Thr Ile Gln Asn Ser Ile Lys Met LysAsn Trp Ser Val 295 300 305 AGG AGA AGT GAC TTC AGA TTC TCT GAA GTT CATGGT GCA GAG AAT TTT 1135 Arg Arg Ser Asp Phe Arg Phe Ser Glu Val His GlyAla Glu Asn Phe 310 315 320 325 ATT CAG CAT AAC CTA CAG ACC TTA AAA AGTAAG ATA TTT GAC AAT GAA 1183 Ile Gln His Asn Leu Gln Thr Leu Lys Ser LysIle Phe Asp Asn Glu 330 335 340 TCT GCT GCC TGA AATAAAACCA TTAGGACTCACTGGGACAGA ACTTTCAAGT 1235 Ser Ala Ala TCCTTCAACT GTGAAAAGTG TCTTTTTGGACAAACTATTT TTCCACCTCC AAAAGAAATT 1295 AACACA 1301 344 amino acids aminoacid linear protein 8 Met Val Ser Val Asn Ser Ser His Cys Phe Tyr AsnAsp Ser Phe Lys 1 5 10 15 Tyr Thr Leu Tyr Gly Cys Met Phe Ser Met ValPhe Val Leu Gly Leu 20 25 30 Ile Ser Asn Cys Val Ala Ile Tyr Ile Phe IleCys Val Leu Lys Val 35 40 45 Arg Asn Glu Thr Thr Thr Tyr Met Ile Asn LeuAla Met Ser Asp Leu 50 55 60 Leu Phe Val Phe Thr Leu Pro Phe Arg Ile PheTyr Phe Thr Thr Arg 65 70 75 80 Asn Trp Pro Phe Gly Asp Leu Leu Cys LysIle Ser Val Met Leu Phe 85 90 95 Tyr Thr Asn Met Tyr Gly Ser Ile Leu PheLeu Thr Cys Ile Ser Val 100 105 110 Asp Arg Phe Leu Ala Ile Val Tyr ProPhe Lys Ser Lys Thr Leu Arg 115 120 125 Thr Lys Arg Asn Ala Lys Ile ValCys Thr Gly Val Trp Leu Thr Val 130 135 140 Ile Gly Gly Ser Ala Pro AlaVal Phe Val Gln Ser Thr His Ser Gln 145 150 155 160 Gly Asn Asn Ala SerGlu Ala Cys Phe Glu Asn Phe Pro Glu Ala Thr 165 170 175 Trp Lys Thr TyrLeu Ser Arg Ile Val Ile Phe Ile Glu Ile Val Gly 180 185 190 Phe Phe IlePro Leu Ile Leu Asn Val Thr Cys Ser Ser Met Val Leu 195 200 205 Lys ThrLeu Thr Lys Pro Val Thr Leu Ser Arg Ser Lys Ile Asn Lys 210 215 220 ThrLys Val Leu Lys Met Ile Phe Val His Leu Ile Ile Phe Cys Phe 225 230 235240 Cys Phe Val Pro Tyr Asn Ile Asn Leu Ile Leu Tyr Ser Leu Val Arg 245250 255 Thr Gln Thr Phe Val Asn Cys Ser Val Val Ala Ala Val Arg Thr Met260 265 270 Tyr Pro Ile Thr Leu Cys Ile Ala Val Ser Asn Cys Cys Phe AspPro 275 280 285 Ile Val Tyr Tyr Phe Thr Ser Asp Thr Ile Gln Asn Ser IleLys Met 290 295 300 Lys Asn Trp Ser Val Arg Arg Ser Asp Phe Arg Phe SerGlu Val His 305 310 315 320 Gly Ala Glu Asn Phe Ile Gln His Asn Leu GlnThr Leu Lys Ser Lys 325 330 335 Ile Phe Asp Asn Glu Ser Ala Ala 340 30BASE PAIRS NUCLEIC ACID SINGLE LINEAR Oligonucleotide 9 GACTAAAGCTTAATGAGTAG TGAAATGGTG 30 31 BASE PAIRS NUCLEIC ACID SINGLE LINEAROligonucleotide 10 GAACTTCTAG ACCCTCAGGG TTGTAAATCA G 31 30 BASE PAIRSNUCLEIC ACID SINGLE LINEAR Oligonucleotide 11 GACTAAAGCT TAATGAGGCCCACATGGGCA 30 32 BASE PAIRS NUCLEIC ACID SINGLE LINEAR Oligonucleotide12 GAACTTCTAG ACGAACTAGT GGATCCCCCC GG 32 30 BASE PAIRS NUCLEIC ACIDSINGLE LINEAR Oligonucleotide 13 GACTAAAGCT TAATGGCGTC TTTCTCTGCT 30 30BASE PAIRS NUCLEIC ACID SINGLE LINEAR Oligonucleotide 14 GAACTTCTAGACTTCACACA GTTGTACTAT 30 30 BASE PAIRS NUCLEIC ACID SINGLE LINEAROligonucleotide 15 GACTAAAGCT TAATGGTAAG CGTTAACAGC 30 31 BASE PAIRSNUCLEIC ACID SINGLE LINEAR Oligonucleotide 16 GAACTTCTAG ACTTCAGGCAGCAGATTCAT T 31 34 BASE PAIRS NUCLEIC ACID SINGLE LINEAR Oligonucleotide17 GTCCAAGCTT GCCACCATGA GTAGTGAAAT GGTG 34 58 BASE PAIRS NUCLEIC ACIDSINGLE LINEAR Oligonucleotide 18 CTAGCTCGAG TCAAGCGTAG TCTGGGACGTCGTATGGGTA GCAGGGTTGT AAATCAGG 58 34 BASE PAIRS NUCLEIC ACID SINGLELINEAR Oligonucleotide 19 GTCCAAGCTT GCCACCATGG TTGGTGGCAC CTGG 34 58BASE PAIRS NUCLEIC ACID SINGLE LINEAR Oligonucleotide 20 CTAGCTCGAGTCAAGCGTAG TCTGGGACGT CGTATGGGTA GCAGTGGATC CCCCGTGC 58 34 BASE PAIRSNUCLEIC ACID SINGLE LINEAR Oligonucleotide 21 GTCCAAGCTT GCCACCATGAACACCACAGT AATG 34 61 BASE PAIRS NUCLEIC ACID SINGLE LINEAROligonucleotide 22 CTAGCTCGAG TCAAGCGTAG TCTGGGACGT CGTATGGGTAGCAAGGGATC CATACAAATG 60 T 61 34 BASE PAIRS NUCLEIC ACID SINGLE LINEAROligonucleotide 23 GTCCAAGCTT GCCACCATGG TAAGCGTTAA CAGC 34 61 BASEPAIRS NUCLEIC ACID SINGLE LINEAR Oligonucleotide 24 CTAGCTCGAGTCAAGCGTAG TCTGGGACGT CGTATGGGTA GCAGGCAGCA GATTCATTGT 60 C 61 30 BASEPAIRS NUCLEIC ACID SINGLE LINEAR Oligonucleotide 25 CGGGATCCCTCCATGAGTAG TGAAATGGTG 30 29 BASE PAIRS NUCLEIC ACID SINGLE LINEAROligonucleotide 26 CGGGATCCCG CTCAGGGTTG TAAATCAGG 29 222 amino acidsamino acid single Not Relevant peptide 27 Phe Phe Leu Ser His Leu AlaIle Val Asp Ile Ala Tyr Ala Cys Asn 1 5 10 15 Thr Val Pro Gln Met LeuVal Asn Leu Leu Asp Pro Val Lys Pro Ile 20 25 30 Ser Tyr Ala Gly Cys MetThr Gln Thr Phe Leu Phe Leu Thr Phe Ala 35 40 45 Ile Thr Glu Cys Leu LeuLeu Val Val Met Ser Tyr Asp Arg Tyr Val 50 55 60 Ala Ile Cys His Pro LeuArg Tyr Ser Ala Ile Met Ser Trp Arg Val 65 70 75 80 Cys Ser Thr Met AlaVal Thr Ser Trp Ile Ile Gly Val Leu Leu Ser 85 90 95 Leu Ile His Leu ValLeu Leu Leu Pro Leu Pro Phe Cys Val Ser Gln 100 105 110 Lys Val Asn HisPhe Phe Cys Glu Ile Thr Ala Ile Leu Lys Leu Ala 115 120 125 Cys Ala AspThr His Leu Asn Glu Thr Met Val Leu Ala Gly Ala Val 130 135 140 Ser ValLeu Val Gly Pro Phe Ser Ser Ile Val Val Ser Tyr Ala Cys 145 150 155 160Ile Leu Gly Ala Ile Leu Lys Ile Gln Ser Glu Glu Gly Gln Arg Lys 165 170175 Ala Phe Ser Thr Cys Ser Ser His Leu Cys Val Val Gly Leu Phe Tyr 180185 190 Gly Thr Ala Ile Val Met Tyr Val Gly Pro Arg His Gly Ser Pro Lys195 200 205 Glu Gln Lys Lys Tyr Leu Leu Leu Phe His Ser Leu Phe Asn 210215 220 381 amino acids amino acid single Not Relevant peptide 28 MetGly Pro Thr Ser Val Pro Leu Val Lys Ala His Arg Ser Ser Val 1 5 10 15Ser Asp Tyr Val Asn Tyr Asp Ile Ile Val Arg His Tyr Asn Tyr Thr 20 25 30Gly Lys Leu Asn Ile Ser Ala Asp Lys Glu Asn Ser Ile Lys Leu Thr 35 40 45Ser Val Val Phe Ile Leu Ile Cys Cys Phe Ile Ile Leu Glu Asn Ile 50 55 60Phe Val Leu Leu Thr Ile Trp Lys Thr Lys Lys Phe His Arg Pro Met 65 70 7580 Tyr Tyr Phe Ile Gly Asn Leu Ala Leu Ser Asp Leu Leu Ala Gly Val 85 9095 Ala Tyr Thr Ala Asn Leu Leu Leu Ser Gly Ala Thr Thr Tyr Lys Leu 100105 110 Thr Pro Ala Gln Trp Phe Leu Arg Glu Gly Ser Met Phe Val Ala Leu115 120 125 Ser Ala Ser Val Phe Ser Leu Leu Ala Ile Ala Ile Glu Arg TyrIle 130 135 140 Thr Met Leu Lys Met Lys Leu His Asn Gly Ser Asn Asn PheArg Leu 145 150 155 160 Phe Leu Leu Ile Ser Ala Cys Trp Val Ile Ser LeuIle Leu Gly Gly 165 170 175 Leu Pro Ile Met Gly Trp Asn Cys Ile Ser AlaLeu Ser Ser Cys Ser 180 185 190 Thr Val Leu Pro Leu Tyr His Lys His TyrIle Leu Phe Cys Thr Thr 195 200 205 Val Phe Thr Leu Leu Leu Leu Ser IleVal Ile Leu Tyr Cys Arg Ile 210 215 220 Tyr Ser Leu Val Arg Thr Arg SerArg Arg Leu Thr Phe Arg Lys Asn 225 230 235 240 Ile Ser Lys Ala Ser ArgSer Ser Glu Asn Val Ala Leu Leu Lys Thr 245 250 255 Val Ile Ile Val LeuSer Val Phe Ile Ala Cys Trp Ala Pro Leu Phe 260 265 270 Ile Leu Leu LeuLeu Asp Val Gly Cys Lys Val Lys Thr Cys Asp Ile 275 280 285 Leu Phe ArgAla Glu Tyr Phe Leu Val Leu Ala Val Leu Asn Ser Gly 290 295 300 Thr AsnPro Ile Ile Tyr Thr Leu Thr Asn Lys Glu Met Arg Arg Ala 305 310 315 320Phe Ile Arg Ile Met Ser Cys Cys Lys Cys Pro Ser Gly Asp Ser Ala 325 330335 Gly Lys Phe Lys Arg Pro Ile Ile Ala Gly Met Glu Phe Ser Arg Ser 340345 350 Lys Ser Asp Asn Ser Ser His Pro Gln Lys Asp Glu Gly Asp Asn Pro355 360 365 Glu Thr Ile Met Ser Ser Gly Asn Val Asn Ser Ser Ser 370 375380 325 amino acids amino acid single Not Relevant peptide 29 Ile AsnSer Thr Ser Thr Gln Pro Pro Asp Glu Ser Cys Ser Gln Asn 1 5 10 15 LeuLeu Ile Thr Gln Gln Ile Ile Pro Val Leu Tyr Cys Met Val Phe 20 25 30 IleAla Gly Ile Leu Leu Asn Gly Val Ser Gly Trp Ile Phe Phe Tyr 35 40 45 ValPro Ser Ser Lys Ser Phe Ile Ile Tyr Leu Lys Asn Ile Val Ile 50 55 60 AlaAsp Phe Val Met Ser Leu Thr Phe Pro Phe Lys Ile Leu Gly Asp 65 70 75 80Ser Gly Leu Gly Pro Trp Gln Leu Asn Val Phe Val Cys Arg Val Ser 85 90 95Ala Val Leu Phe Tyr Val Asn Met Tyr Val Ser Ile Val Phe Phe Gly 100 105110 Leu Ile Ser Phe Asp Arg Tyr Tyr Lys Ile Val Lys Pro Leu Trp Thr 115120 125 Ser Phe Ile Gln Ser Val Ser Tyr Ser Lys Leu Leu Ser Val Ile Val130 135 140 Trp Met Leu Met Leu Leu Leu Ala Val Pro Asn Ile Ile Leu ThrAsn 145 150 155 160 Gln Ser Val Arg Glu Val Thr Gln Ile Lys Cys Ile GluLeu Lys Ser 165 170 175 Glu Leu Gly Arg Lys Trp His Lys Ala Ser Asn TyrIle Phe Val Ala 180 185 190 Ile Phe Trp Ile Val Phe Leu Leu Leu Ile ValPhe Tyr Thr Ala Ile 195 200 205 Thr Lys Lys Ile Phe Lys Ser His Leu LysSer Ser Arg Asn Ser Thr 210 215 220 Ser Val Lys Lys Lys Ser Ser Arg AsnIle Phe Ser Ile Val Phe Val 225 230 235 240 Phe Phe Val Cys Phe Val ProTyr His Ile Ala Arg Ile Pro Tyr Thr 245 250 255 Lys Ser Gln Thr Glu AlaHis Tyr Ser Cys Gln Ser Lys Glu Ile Leu 260 265 270 Arg Tyr Met Lys GluPhe Thr Leu Leu Leu Ser Ala Ala Asn Val Cys 275 280 285 Leu Asp Pro IleIle Tyr Phe Phe Leu Cys Gln Pro Phe Arg Glu Ile 290 295 300 Leu Cys LysLys Leu His Ile Pro Leu Lys Ala Gln Asn Asp Leu Asp 305 310 315 320 IleSer Arg Ile Lys 325 302 amino acids amino acid single Not Relevantpeptide 30 Ser Ser Asn Cys Ser Thr Glu Asp Ser Phe Lys Tyr Thr Leu TyrGly 1 5 10 15 Cys Val Phe Ser Met Val Phe Val Leu Gly Leu Ile Ala AsnCys Val 20 25 30 Ala Ile Tyr Ile Phe Thr Phe Thr Leu Lys Val Arg Asn GluThr Thr 35 40 45 Thr Tyr Met Leu Met Leu Ala Ile Ser Asp Leu Leu Phe ValPhe Thr 50 55 60 Leu Pro Phe Arg Ile Tyr Tyr Phe Val Val Arg Asn Trp ProPhe Gly 65 70 75 80 Asp Val Leu Cys Lys Ile Ser Val Thr Leu Phe Tyr ThrAsn Met Tyr 85 90 95 Gly Ser Ile Leu Phe Leu Thr Cys Ile Ser Val Asp ArgPhe Leu Ala 100 105 110 Ile Val His Pro Phe Arg Ser Lys Thr Leu Arg ThrLys Arg Asn Ala 115 120 125 Arg Ile Val Cys Val Ala Val Trp Ile Thr ValLeu Ala Gly Ser Thr 130 135 140 Pro Ala Ser Phe Phe Gln Ser Thr Asn ArgGln Asn Asn Thr Glu Gln 145 150 155 160 Arg Thr Cys Phe Glu Asn Phe ProGlu Ser Thr Trp Lys Thr Tyr Leu 165 170 175 Ser Arg Ile Val Ile Phe IleGlu Ile Val Gly Phe Phe Ile Pro Leu 180 185 190 Ile Leu Asn Val Thr CysSer Thr Met Val Leu Arg Thr Leu Asn Lys 195 200 205 Pro Leu Thr Leu SerArg Asn Lys Leu Ser Lys Lys Lys Val Leu Lys 210 215 220 Met Ile Phe ValHis Leu Val Ile Phe Cys Phe Cys Phe Val Pro Tyr 225 230 235 240 Asn IleThr Leu Ile Leu Tyr Ser Leu Met Arg Thr Gln Thr Trp Ile 245 250 255 AsnCys Ser Val Val Thr Ala Val Arg Thr Met Tyr Pro Val Thr Leu 260 265 270Cys Ile Ala Val Ser Asn Cys Cys Phe Asp Pro Ile Val Tyr Tyr Phe 275 280285 Thr Ser Asp Thr Asn Ser Glu Leu Asp Lys Lys Gln Gln Val 290 295 300

What is claimed is:
 1. An isolated polynucleotide comprising a memberselected from the group consisting of: (a) a polynucleotide encoding thepolypeptide as set forth in SEQ ID NO: 2; (b) a polynucleotide encodingthe polypeptide as set forth in SEQ ID NO: 4; (c) a polynucleotideencoding the polypeptide as set forth in SEQ ID NO: 6; (d) apolynucleotide encoding the polypeptide as set forth in SEQ ID NO: 8;(e) a polynucleotide capable of hybridizing to and which is at least 70%identical to the polynucleotide of (a), (b), (c) or (d); and (f) apolynucleotide fragment of the polynucleotide of (a), (b), (c), (d) or(e).
 2. The polynucleotide of claim 1 wherein the polynucleotide is DNA.3. An isolated polynucleotide comprising a member selected from thegroup consisting of: (a) a polynucleotide which encodes a maturepolypeptide encoded by the DNA contained in ATCC Deposit No. 75981; (b)a polynucleotide which encodes a mature polypeptide encoded by the DNAcontained in ATCC Deposit No. 75983; (c) a polynucleotide which encodesa mature polypeptide encoded by the DNA contained in ATCC Deposit No.75976; (d) a polynucleotide which encodes a mature polypeptide encodedby the DNA contained in ATCC Deposit No. 75979; (e) a polynucleotidecapable of hybridizing to and which is at least 70% identical to thepolynucleotide of (a), (b), (c) or (d); and (f) a polynucleotidefragment of the polynucleotide of (a), (b), (c), (d) or (e).
 4. A vectorcontaining the DNA of claim
 2. 5. A host cell genetically engineeredwith the vector of claim
 4. 6. A process for producing a polypeptidecomprising: expressing from the host cell of claim 5 the polypeptideencoded by said DNA.
 7. A process for producing cells capable ofexpressing a polypeptide comprising transforming or transfecting thecells with the vector of claim
 4. 8. A polypeptide selected from thegroup consisting of: (i) a polypeptide having the deduced amino acidsequence of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6 and SEQ ID No. 8and fragments, analogs and derivatives thereof, (ii) a polypeptideencoded by the cDNA of ATCC Deposit No. 75981, ATCC Deposit No. 75983,ATCC Deposit No. 75976 and ATCC Deposit No. 75979 and fragments, analogsand derivatives of said polypeptide.
 9. An antibody against thepolypeptide of claim
 8. 10. A compound which activates the thepolypeptide of claim
 8. 11. A compound which inhibits activation of thepolypeptide of claim
 8. 12. A method for the treatment of a patienthaving need to activate a G-protein coupled receptor comprising:administering to the patient a therapeutically effective amount of thecompound of claim
 10. 13. A method for the treatment of a patient havingneed to inhibit activation of a G-protein coupled receptor comprising:administering to the patient a therapeutically effective amount of thecompound of claim
 11. 14. The polypeptide of claim 8 wherein thepolypeptide is a soluble fragment of the G-protein coupled receptor andis capable of binding a ligand for the receptor.
 15. A process foridentifying antagonists and agonists to the polypeptide of claim 8comprising: contacting a cell which expresses a G-protein coupledreceptor with a known receptor ligand and a compound to be screened; anddetermining if the compound inhibits or enhances activation of thereceptor.
 16. A method for diagnosing a disease or a susceptibility to adisease comprising: detecting a mutation in the nucleic acid sequenceencoding the polypeptide of claim 8 in a sample derived from a host. 17.A diagnostic process comprising: analyzing for the presence of thepolypeptide of claim 14 in a sample derived from a host.
 18. Thepolynucleotide of claim 2 which encodes the polypeptide as set forth inSEQ ID NO:
 2. 19. The polynucleotide of claim 2 which encodes thepolypeptide as set forth in SEQ ID NO:
 4. 20. The polynucleotide ofclaim 2 which encodes the polypeptide as set forth in SEQ ID NO: 6.